NF-AT polypeptides and polynucleotides

ABSTRACT

The invention provides novel polypeptides which are associated with the transcription complex NF-AT, polynucleotides encoding such polypeptides, antibodies which are reactive with such polypeptides, polynucleotide hybridization probes and PCR amplification probes for detecting polynucleotides which encode such polypeptides, transgenes which encode such polypeptides, homologous targeting constructs that encode such polypeptides and/or homologously integrate in or near endogenous genes encoding such polypeptides, nonhuman transgenic animals which comprise functionally disrupted endogenous genes that normally encode such polypeptides, and transgenic nonhuman animals which comprise transgenes encoding such polypeptides. The invention also provides methods for detecting T cells (including activated T cells) in a cellular sample, methods for treating hyperactive or hypoactive T cell conditions, methods for screening for immunomodulatory agents, methods for diagnostic staging of lymphocyte differentiation, methods for producing NF-AT proteins for use as research or diagnostic reagents, methods for producing antibodies reactive with the novel polypeptides, and methods for producing transgenic nonhuman animals.

This application is a continuation application of Ser. No. 08/260,174filed on Jun. 13, 1994, now U.S. Pat. No. 6,197,925 which is acontinuation-in-part of U.S. Ser. No. 08/124,981 filed Sep. 20, 1993(now U.S. Pat. No. 5,837,840).

STATEMENT OF RIGHTS

This invention was made in the course of work supported by the U.S.Government and Howard Hughes Medical Institute, which may have certainrights in this invention.

FIELD OF THE INVENTION

The invention provides novel polypeptides which are associated with thetranscription complex NF-AT, polynucleotides encoding such polypeptides,antibodies which are reactive with such polypeptides, polynucleotidehybridization probes and PCR amplification probes for detectingpolynucleotides which encode such polypeptides, transgenes which encodesuch polypeptides, homologous targeting constructs that encode suchpolypeptides and/or homologously integrate in or near endogenous genesencoding such polypeptides, nonhuman transgenic animals which comprisefunctionally disrupted endogenous genes that normally encode suchpolypeptides, and transgenic nonhuman animals which comprise transgenesencoding such polypeptides. The invention also provides methods fordetecting T cells (including activated T cells) in a cellular sample,methods for treating hyperactive or hypoactive T cell conditions,methods for screening for immunomodulatory agents, methods fordiagnostic staging of lymphocyte differentiation, methods for producingNF-AT proteins for use as research or diagnostic reagents, methods forproducing antibodies reactive with the novel polypeptides, and methodsfor producing transgenic nonhuman animals.

BACKGROUND OF THE INVENTION

The immune response is coordinated by the actions of cytokines producedfrom activated T lymphocytes. The precursors for most T lymphocytesarise in the bone marrow and migrate to the thymus where theydifferentiate and express receptors capable of interacting with antigen.These differentiated T lymphocytes then migrate to the peripherallymphoid organs where they remain quiescent until they come in contactwith the cognate antigen. The interaction of antigen with the antigenreceptor on T lymphocytes initiates an ordered series of pleiotropicchanges; a process denoted as T lymphocyte activation. T lymphocyteactivation is a 7 to 10 day process that results in cell division andthe acquisition of immunological functions such as cytotoxicity and theproduction of lymphokines that induce antibody production by Blymphocytes and control the growth and differentiation of granulocyteand macrophage precursors. The cytokines produced by activated Tlymphocytes act upon other cells of the immune system to coordinatetheir behavior and bring about an effective immune response.

The initiation of T lymphocyte activation requires a complex interactionof the antigen receptor with the combination of antigen andself-histocompatibility molecules on the surface of antigen-presentingcells. T lymphocytes may also be activated by relatively simple stimulisuch as the combination of a calcium ionophore (e.g., ionomycin) and anactivator of protein kinase C, such as phorbol myristate acetate (PMA).Several lectins, including phytohemagglutinin (PHA) may also be used toactivate T cells (Nowell (1960) Cancer Res. 20: 462).

T lymphocyte activation involves the specific regulation of particularsubsets of genes. The transcriptional regulation characteristic of Tcell activation begins minutes after the antigen encounter and continuesuntil at least 10 days later. The T lymphocyte activation genes can begrouped according to the time after stimulation at which each gene istranscribed. Early genes are the first subset of T lymphocyte activationgenes that is expressed during the activation process. Expression of theearly genes triggers the transcriptional modulation of subsequent genesin the activation pathway. Because of the critical role of the Tlymphocyte in the immune response, agents that interfere with expressionof the early activation genes, such as cyclosporin A and FK506, areeffective immunosuppressants.

Transcription of the early genes requires the presence of specifictranscription factors, such as NF-AT, which in turn are regulatedthrough interactions with the antigen receptor. These transcriptionfactors are proteins which act through enhancer and promoter elementsnear the early activation genes to modulate the rate of transcription ofthese genes. Many of these transcription factors reversibly bind tospecific DNA sequences located in and near enhancer elements.

The interleukin-2 (IL-2) gene is a paradigmatic early activation gene.The IL-2 gene product plays a critical role in T lymphocyteproliferation and differentiation. The IL-2 gene is transcriptionallyactive only in T cells that have been stimulated through the antigenreceptor or its associated molecules (Cantrell and Smith (1984) Science224: 1312). The transcriptional induction of IL-2 in activated Tlymphocytes is amediated by a typical early gene transcriptionalenhancer that extends from 325 basepairs upstream of the transcriptionalstart site for the IL-2 gene (Durand et al. (1988) Mol. Cell. Biol. 8:1715). Other genes known to contain NF-AT recognition sites in theirregulatory regions include: γ-interferon, IL-4, GM-CSF, and others. Thisregion, which is referred to herein as the IL-2 enhancer, has been usedextensively to dissect the requirements for T lymphocyte activation. Anarray of transcription factors, including NF-AT, NFkb, AP1, Oct-1, and anewly identified protein that associates with Oct-1 called OAP-40, bindto sequences in this region (Ullman et al. (1991) Science 254: 558).These different transcription factors act together to integrate thecomplex requirements for T lymphocyte activation.

Among the group of transcription factors mentioned above, the presenceof NF-AT is characteristic of the transcription events involving earlyactivation genes, in that its recognition sequence is able to enhancetranscription of linked heterologous genes in activated T cells oftransgenic animals (Verweij et al. (1990) J. Biol. Chem. 265: 15788).The NF-AT sequence element is also the only known transcriptionalelement in the IL-2 enhancer that has no stimulatory effect ontranscription in the absence of physiologic activation of the Tlymphocyte through the antigen receptor or through treatment of T cellswith the combination of ionomycin and PMA. For example, the NF-ATelement enhances transcription of linked sequences in T lymphocyteswhich have had proper presentation of specific antigen by MHC-matchedantigen presenting cells or have been stimulated with the combination ofionomycin/PMA, but not in unstimulated T lymphocytes (Durand et al.(1988) op.cit; Shaw et al. (1988) op.cit; Karttunen and Shastri (1991)Proc. Natl. Acad. Sci. USA 88: 3972; Verweij et al. (1990) op.cit).Moreover, the NF-AT sequence element naturally enhances transcription ofthe IL-2 gene only in activated T lymphocytes.

Other elements within the IL-2 enhancer, for example, the NFkb site orthe AP-1 site, activate transcription in response to less specificstimuli, such as tumor necrosis factor a or simply PMA by itself. Thesecompounds do not by themselves activate transcription of the IL-2 geneand other early activation genes, and do not lead to T lymphocyteactivation.

Such observations indicate that the expression of certain early genes,such as the interleukin-2 gene may be regulated by the protein complexNF-AT. Data have also indicated that a selective genetic deficiency ofNF-AT produces severe combined immunodeficiency (SCID) (Chatilla et al.(1989) New Engl. J. Med. 320: 696).

One of the functional sequences in the IL-2 enhancer is a binding sitefor a multimeric protein complex, designated NF-AT (nuclear factor ofactivated T lymphocytes), that functions as a transcriptional regulatorof IL-2, IL-4, and other early activation genes (Shaw et al. (1988)Science 241: 202). The NF-AT transcription complex is formed subsequentto a signal from the antigen receptor. Enhancement of transcription ofgenes adjacent to the NF-AT recognition site requires that the NF-ATcomplex bind to the recognition site (Shaw et al. (1988) op.cit).Although the molecular makeup of NF-AT is not fully defined, studieshave reported that NF-AT can be reconstituted from a ubiquitous nuclearcomponent that requires protein synthesis for induction and a Tcell-specific constitutive cytoplasmic component, designated NF-AT_(c)(Flanagan et al. (1991) Nature 352: 803). This cytoplasmic component,NF-AT_(c), associates with the nucleus in response to calcium signallingin a manner that is inhibited by the immunosuppressive drugs cyclosporinA (CsA) and FK506. The nuclear component of NF-AT can be induced withPMA, is not sensitive to CsA or FK506, and can be seen in cells of non-Tcell origin such as HeLa and Cos.

Northrop et al. (1993) J. Biol. Chem. 268: 2917 report that the nuclearcomponent of NF-AT contains the phorbol ester-inducible transcriptionfactor, AP1 (Jun/Fos), and show that antisera to Fos (a component ofAP1) inhibits NF-AT binding to DNA containing a binding site for AP-1.Moreover, Northrop et al. show that NF-AT DNA binding can bereconstituted in vitro using semi-purified AP-1 proteins mixed withcytosol from T lymphocytes, presumably containing NF-AT_(c). Northrop etal. also report partial purification of NF-AT_(c) and report a molecularmass range of approximately 94 to 116 kD as estimated bySDS-polyacrylamide gel electrophoresis.

As noted above, cyclosporin A (CsA) and FK506 are capable of acting asimmunosuppressants. These agents inhibit T and B cell activation, mastcell degranulation, and other processes essential to an effective immuneresponse (Borel et al. (1976) Agents Actions 6: 468; Sung et al. (1988)J. Exp. Med. 168: 1539; Gao et al. Nature 336: 176). In T lymphocytes,these drugs disrupt a step in the signal transduction pathway(s) throughwhich the binding of antigen to the T cell antigen receptor producesenhanced transcription of specific cytokine genes involved in thecoordination of the immune response. Thus, these agents prevent Tlymphocyte activation (Crabtree et al. (1989) Science 243: 355;Schreiber et al. (1989) Science 251:283; Hohman & Hutlsch (1990) NewBiol. 2: 663) and act as immunosuppressants.

Putative intracellular receptors for FK506 and CsA have been describedand found to be cis-trans prolyl isomerases (Fischer & Bang (1985)Biochim. Biophys. Acta 828: 39; Fischer et al. Nature 337: 476;Handschumacher et al. (1984) Science 226: 544; Lang & Schmid (1988)Nature 331: 453; Standaert et al. (1990) Nature 346: 671). Binding ofthe drugs inhibits isomerase activity; however, studies with otherprolyl isomerase inhibitors (Bierer et al. (1990) Science 250: 556) andanalysis of cyclosporin-resistant mutants in yeast suggest that theprevention of T lymphocyte activation results from formation of aninhibitory complex involving the drug and the isomerase (Bierer et al.(1990) Proc. Natl. Acad. Sci. U.S.A. 87: 9231; Tropschug et al. (1989)Nature 342: 953), and not from inhibition of the isomerase activity perse. CsA and FK-506 prevent T cell proliferation by inhibiting acalcium-dependent signalling event required for the induction ofinterleukin-2 transcription.

Calcineurin, a calmodulin-dependent protein phosphatase which occurs invarious isoforms, has been identified as a critical component of T cellactivation through the signal transduction pathway leading totranscriptional activation of NF-AT-dependent genes, such as lymphokinegenes (Liu et al. (1991) Cell 66: 807; Clipstone and Crabtree (1992)Nature 357: 695; O'Keefe et al. (1992) Nature 357: 692)

Transcriptional enhancement involving NF-AT recognition sequences iscompletely blocked in T cells treated with efficacious concentrations ofcyclosporin A or FK506, with little or no specific effect ontranscriptional enhancement involving recognition sites for othertranscription factors, such as AP-1 and NF-κB (Shaw et al.(1988) op.cit;Emmel et al. (1989) Science 246: 1617; Mattila et al. (1990) EMBO J. 9:4425). This blockage can be overcome, at least partially, by theexpression of hyperphysiolgical amounts of calcineurin (Clipstone andCrabtree (1992) op.cit.).

Unfortunately, while both cyclosporin A and FK506 are potentimmunosuppressive agents, both drugs possess detrimental properties. Forexample, cyclosporin elicits adverse reactions. including renaldysfunction, tremors, nausea and hypertension. Indeed, for many yearsresearchers have attempted to develop superior replacements, with FK506being the most recent candidate. However, without understanding themechanisms by which cyclosporin (or FK506) functions at theintracellular level, developing improved immunosuppressants representsan extremely difficult research effort with a limited likelihood ofsuccess.

Thus, there exists a significant need to understand the functional basisof T cell activation involving NF-AT, particularly with regard to themechanism by which these immunosuppresants such as CsA and FK506 inhibittranscription of the early activation genes. With such knowledge,improved assays for screening drug candidates would be feasible, whichcould in turn dramatically enhance the search process. Modulation of theimmune system, especially modulation of T cell activation, also may beeffected by directly altering the amount or activity of NF-AT. Thepresent invention fulfills these and other needs.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as anadmission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

The present invention provides several novel methods and compositionsfor modulating the immune response and for screening for modulators ofthe immune response. These methods utilize polynucleotide sequencesencoding NF-AT_(c) recombinant proteins and complementarypolynucleotides which are substantially identical to NF-AT_(c)polynucleotide sequences.

In one aspect of the invention, NF-AT_(c) polypeptides and compositionsthereof are provided. NF-AT_(c) polypeptides comprise polypeptidesequences which are substantially identical to a sequence shown in FIG.1 or a cognate NF-AT_(c) gene sequence.

Nucleic acid sequences encoding NF-AT_(c) are provided. Thecharacteristics of the cloned sequences are given, including thenucleotide and predicted amino acid sequence in FIG. 1. Polynucleotidescomprising these sequences can serve as templates for the recombinantexpression of quantities of NF-AT_(c) polypeptides, such as humanNF-AT_(c) and murine NF-AT_(c). Polynucleotides comprising thesesequences can also serve as probes for nucleic acid hybridization todetect the transcription and mRNA abundance of NF-AT_(c) mRNA inindividual lymphocytes (or other cell types) by in situ hybridization,and in specific lymphocyte populations by Northern blot analysis and/orby in situ hybridization (Alwine et al. (1977) Proc. Natl. Acad. Sci.U.S.A. 74: 5350) and/or PCR amplification and/or LCR detection. Suchrecombinant polypeptides and nucleic acid hybridization probes haveutility for in vitro screening methods for immunomodulatory agents andfor diagnosis and treatment of pathological conditions and geneticdiseases, such as transplant rejection reactions, T cell-mediated immuneresponses, lymphocytic leukemias (e.g., T cell leukemia or lymphoma)wherein NF-AT activity contributes to disease processes, autoimmunedisease, arthritis, and the like.

In one embodiment, candidate immunomodulatory agents are identified bytheir ability to block the binding of a NF-AT_(c) polypeptide to othercomponents of NF-AT (e.g., AP-1) and/or to block the binding of NF-AT toDNA having an NF-AT recognition site. The DNA preferably includes one ormore NF-AT binding sites at which a NF-AT protein complex specificallybinds. One means for detecting binding, of a NF-AT protein comprisingNF-AT_(c) to DNA is to immobilize the DNA, such as by covalent ornoncovalent chemical linkage to a solid support, and to contact theimmobilized DNA with a NF-AT protein complex comprising a NF-AT_(c)polypeptide that has been labeled with a detectable marker (e.g., byincorporation of radiolabeled amino acid). Such contacting is typicallyperformed in aqueous conditions which permit binding of a NF-AT proteinto a target DNA containing a NF-AT binding sequence. Binding of thelabeled NF-AT to the immobilized DNA is measured by determining theextent to which the labeled NF-AT_(c) polypeptide is immobilized as aresult of a specific binding interaction. Such specific binding may bereversible, or may be optionally irreversible if a cross-linking agentis added in appropriate experimental conditions.

In one aspect, candidate immunomodulatory agents are identified as beingagents capable of inhibiting (or enhancing) intermolecular bindingbetween NF-AT_(c) and other polypeptides which compriss a NF-AT complex(e.g., AP1, JunB, etc.). The invention provides methods and compositionsfor screening libraires of agents for the capacity to interfere withbinding of NF-AT_(c) to other NF-AT polypeptide species under aqueousbinding conditions. Typically, at least either NF-AT_(c) and/or anotherNF-AT polypeptide species is labeled with a detectable label andintermolecular binding between NF-AT_(c) and other NF-AT polypeptidespecies is detected by the amount of labeled species captured in NF-ATcomplexes and the like.

The invention also provides antisense polynucleotides complementary toNF-AT_(c) sequences which are employed to inhibit transcription and/ortranslation of the cognate mRNA species and thereby effect a reductionin the amount of the respective NF-AT_(c) protein in a cell (e.g., a Tlymphocyte of a patient). Such antisense polynucleotides can function asimmunomodulatory drugs by inhibiting the formation of NF-AT proteinrequired for T cell activation.

In a variation of the invention, polynucleotides of the invention areemployed for diagnosis of pathological conditions or genetic diseasethat involve T cell neoplasms or T cell hyperfunction of hypofunction,and more specifically conditions and diseases that involve alterationsin the structure or abundance of NF-AT_(c) polypeptide, NF-AT_(c)polynucleotide sequence, or structure of the NF-AT_(c) gene or flankingregion(s).

The invention also provides antibodies which bind to NF-AT_(c) with anaffinity of about at least 1×10⁷ M⁻¹ and which lack specific highaffinity binding for other proteins present in activated T cells. Suchantibodies can be used as diagnostic reagents to identify T cells (e.g.,activatable T cells) in a cellular sample from a patient (e.g., alymphocyte sample, a solid tissue biopsy) as being cells which containan increased amount of NF-AT_(c) protein determined by standardizationof the assay to be diagnostic for activated T cells. Frequently,anti-NF-AT_(c) antibodies are included as diagnostic reagents forimmunohistopathology staining of cellular samples in situ. Additionally,anti-NF-AT_(c) antibodies may be used therapeutically by targeteddelivery to T cells (e.g., by cationization or byliposome/immunoliposome delivery).

The invention also provides NF-AT_(c) polynucleotide probes fordiagnosis of neoplasia or immune status by detection of NF-AT_(c) mRNAin cells explanted from a patient, or detection of a pathognomonicNF-AT_(c) allele (e.g., by RFLP or allele-specific PCR analysis). Apathognomonic NF-AT_(c) allele is an allele which is statisticallycorrelated with the presence of a predetermined disease or propensity todevelop a disease. Typically, the detection will be by in situhybridization using a labeled (e.g., ³²p, ³⁵S, ¹⁴C, ³H, fluorescent,biotinylated, digoxigeninylated) NF-AT_(c) polynucleotide, althoughNorthern blotting, dot blotting, or solution hybridization on bulk RNAor poly A⁺ RNA isolated from a cell sample may be used, as may PCRamplification using NF-AT_(c)-specific primers. Cells which contain anincreased amount of NF-AT_(c) mRNA as compared to standard controlvalues for cells or cell types other than activated T cells oractivatable T cells will be thereby identified as activated T cells oractivatable T cells. Similarly, the detection of pathognomonicrearrangements or amplification of the NF-AT_(c) locus or closely linkedloci in a cell sample will identify the presence of a pathologicalcondition or a predisposition to developing a pathological condition(e.g., cancer, genetic disease).

The present invention also provides a method for diagnosing T cellhypofunction of hyperfunction in a human patient, wherein a diagnosticassay (e.g., immunchistochemical staining of fixed lymphocytic cells byan antibody that specifically binds human NF-AT_(c)) is used todetermine if a predetermined pathognomonic concentration of NF-AT_(c)protein or NF-AT_(c) mRNA is present in a biological sample from a humanpatient; if the assay indicates the presence of NF-AT_(c) protein orNF-AT_(c) mRNA at or above such predetermined pathognomonicconcentration, the patient is diagnosed as having T cell hyperfunctionor hypofunction condition, or transplant rejection and the like.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E (SEQ ID Nos. 45 and 38 (or 46)) show the nucleotide sequenceof the human NF-AT_(c) cDNA and the deduced amino acid sequence.Nucleotide sequence and complete predicted amino acid sequence ofNF-AT_(c) from human T lymphocytes. Bovine peptides (sequences notcompletely conserved) identified in the predicted human sequence areunderlined. The cDNA ends in a canonical polyadenylation signal and apoly A tail.

FIG. 2 shows the expression of NF-AT_(c) protein in T cells (Jurkat) andnon-T cells (Cos).

FIG. 3A and 3B show that the NF-AT_(c) cDNA clone encodes a protein thatactivates transcription from an NF-AT site and is capable of activatingthe IL-2 promoter in non-T cells.

FIGS. 4A-4C (SEQ ID NOS:47-52) shows homology between NF-AT_(c),NF-AT_(p), and Rel family members. The protein sequences of murineNF-AT_(p) and the Rel proteins Dorsal (the Drosophila axis-determiningprotein), human c-Rel, NF-κB p50, and NF-κB p65 are aligned to thesequence of NF-AT_(c). Numbering is with respect to NF-AT_(c). Identityto NF-AT_(c), open boxes; similarity in known residue function orstructure, shaded areas.. Stars indicate regions in which NF-AT_(c)has: 1) a charge reversal relative to the majority of other Relproteins, or has 2) replaced a potential salt bridge residue with ahistidine or other chelating residue. Lower portion shows a schematic ofNF-AT_(c) and NF-AT_(p).

FIGS. 5A-5C. FIG. 5A Ribonuclease protection for human NF-AT_(c) withRNA from Jurkat cells (lanes 1-6) or Hela cells (lane 7). The expectedspecific ribonuclease-resistant fragment is 304 nucleotides (arrow).Hela cells were non-stimulated. Jurkat cells were either non-stimulatedor stimulated with 20 ng/ml PMA and 2 uM ionomycin for 3 hours, plus orminus 100 ng/ml CsA added at the indicated times after stimulation. FIG.5B: RNA from the following human cells: KJ (preB cell ALL), JD-1 (B celllineage ALL), K562 (erythroleukemia cell line), CML (bone marrow cellsfrom a patient with a myeloid leukemia), human muscle tissue, Hep G2(liver cell line), HPB ALL (T cell line, nonstimulated or stimulatedwith 2 ug/mi PHA and 50 ng/ml PMA for 30 minutes), and Hela cellsanalyzed by ribonuclease protection. A longer exposure of this gelindicates that the K562 cell line contains a small amount of NF-AT_(c)transcript. FIG. 5C: NF-AT_(c) (upper panel) and NF-AT_(p) (lower panel)mRNA expression in mouse tissues and a skin tumor derived from NF-AT-Tagtransgenic mice (Verweij et al. (1990) J. Biol. Chem 265: 15788-15795).Cells were either non-stimulated or stimulated with 20 ng/ml PMA and 2uM ionomycin for 3 hours. RNA was measured by quantiative ribonucleaseprotection using murine cDNA probes. The predicted size of the fragmenthomologous to the probe is indicated by the arrows.

FIGS 6A-6D. FIG. 6A: Cos cells and Jurkat cells were transfected withreporter constructs for NF-AT or HNF-1 (β28). Co-transfected expressionvectors for NF-AT_(c) (+NF-AT) or HNF-1α(+HNF-1) were included whereindicated, otherwise empty pBJ5 vector was included. Cells werestimulated as indicated: PMA, P+I (PMA plus ionomycin). FIG. 6B. Coscells were transfected with IL-2 luciferase and with expression vectorsas in FIG. A. Stimulations were as in a. Data in FIGS 6A and 6B areexpressed as fold induction of luciferase activity over nonstimulatedvalue with empty pBJ5 vector. Bars represent mean and range of 2-3independent transfections. FIG. 6C. Expression of NF-AT_(c) in Cos cellsgives rise to specific DNA binding activity. Gel mobility shifts usingnuclear extracts from Cos cells transfected with pBJ5 (lanes 1 and 3),with NF-AT_(c) (lanes 2 and 4-7), from non-transfected jurkat cells(lanes 8-11) or using cytosols from pBJ5- or NF-AT_(c)-transfected Coscells (lanes 12-13, 15-16) combined with Hela nuclear extract (lanes15-16). Lane 14, Hela nuclear extract alone. Labeled AP-1 (lanes 1-2) orNF-AT (lanes 3-16) probes and cold competitor oligonucleotides areindicated. Arrows indicate specific AP-1 and NF-AT complexes. FIG. 6D:Antisera induced supershift of NF-AT. NF-AT and AP-1 gel mobility shiftsusing nuclear extracts from stimulated Jurkat cells or murinethymocytes. Either no antisera, preimmune, or one of two differentimmune antisera was included as indicated. Arrows indicate specificNF-AT or AP1 complexes or supershifted NF-AT complexes (*).

FIG. 7 shows dominant-negative NF-AT_(c). Jurkat Tag cells weretransfected with vector plasmid (control) or with the dominant negativeNF-AT_(c) plasmid, plus the indicated secreted alkaline phosphatasereporter plasmid. Transfected cells were transferred to fresh culturemedium 24 hours after transfection and secreted alkaline phosphataseactivity was measured (Clipstone and Crabtree (1992) Nature 357:695-698) 16 to 24 hours later, after stimulation with 1 uM ionomycinplus 20 ng/ml PMA (NF-AT and IL-2 reporters), 20 ng/ml PMA alone (APIreporter) or no stimulation (RSV reporter). Bars indicate, secretedalkaline phosphatase activity from cells transfected with the dominantnegative NF-AT_(c) as a percentage of the activity from cellstransfected in parallel with control plasmid, and represent dataobtained from (n) independent transfections. The dominant negativeNF-AT_(c) consists of a carboxy terminal truncation of the epitopetagged NF-AT_(c) expression plasmid extending to the PvuII site at aminoacid 463.

FIG. 8 shows changes in mobility of epitope tagged NF-AT_(c) expressedin Jurkat cells. Cells were transfected with NF-AT_(c) as in FIG. 2 andstimulated as shown for 2 hrs plus or minus 100 ng/ml CsA. Whole celllysates were analyzed by western blotting as in FIG. 2.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage (Immunology—A Synthesis, 2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991), which is incorporated herein by reference).Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention. Similarly,unless specified otherwise, the lefthand end of single-strandedpolynucleotide sequences is the 5′ end; the lefthand direction ofdouble-stranded polynucleotide sequences is referred to as the 5′direction. The direction of 5′ to 3′ addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5′ to the5′ end of the RNA transcript are referred to as “upstream sequences”;sequence regions on the DNA strand having the same sequence as the RNAand which are 3′ to the 3′ end of the RNA transcript are referred to as“downstream sequences”.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparision; a reference sequence may bea subset of a larger sequence, for example, as a segment of afull-length cDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence of FIG. 1, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2: 482, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.(U.S.A.) 85: 2444, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by inspection, and the best alignment (i.e., resulting in thehighest percentage of homology over the comparison window) generated bythe various methods is selected. The term “sequence identity” means thattwo polynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparision (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 85 percentsequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence, for example, as a segment of thefull-length human NF-AT_(c) polynucleotide sequence shown in FIG. 1 orthe full-length murine or bovine NF-AT_(c) cDNA sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “NF-AT_(c) native protein” and “full-length NF-AT_(c) protein”as used herein refers to a a naturally-occurring NF-AT_(c) polypeptidecorresponding to the deduced amino acid sequence shown in FIG. 1 orcorresponding to the deduced amino acid sequence of a cognatefull-length cDNA. Also for example, a native NF-AT_(c) protein presentin naturally-occurring lymphocytes which express the NF-AT_(c) gene areconsidered full-length NF-AT_(c) proteins.

The term “NF-AT_(c) fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the NF-AT_(c) sequence deduced from a full-length cDNAsequence (e.g., the cDNA sequence shown in FIG. 1). NF-AT_(c) fragmentstypically are at least 14 amino acids long, preferably at least 20 aminoacids long, usually at least 50 amino acids long or longer.

The term “NF-AT_(c) analog” as used herein refers to polypeptides whichare comprised of a segment of at least 25 amino acids that hassubstantial identity to a portion of the deduced amino acid sequenceshown in FIG. 1, and which has at least one of the following properties:(1) binding to other NF-AT proteins (e.g., AP-1) under suitable bindingconditions, or (2) ability to localize to the nucleus upon T cellactivation. Typically, NF-AT_(c) analog polypeptides comprise aconservative amino acid substitution (or addition or deletion) withrespect to the naturally-occurring sequence. NF-AT_(c) analogs typicallyare at least 20 amino acids long, preferably at least 50 amino acidslong or longer, most usually being as long as full-lengthnaturally-occurring NF-AT_(c) (e.g., as shown in FIG. 1). Some NF-AT_(c)analogs may lack biological activity but may still be employed forvarious uses, such as for raising antibodies to NF-AT_(c) epitopes, asan immunological reagent to detect and/or purify α-NF-AT_(c) antibodiesby affinity chromatography, or as a competitive or noncompetitiveagonist, antagonist, or partial agonist of native NF-AT_(c) proteinfunction.

The term “NF-AT_(c) polypeptide” is used herein as a generic term torefer to native protein, fragments, or analogs of NF-AT_(c). Hence,native NF-AT_(c), fragments of NF-AT_(c), and analogs of NF-AT_(c) arespecies of the NF-AT_(c) polypeptide genus. Preferred NF-AT_(c)polypeptides include: the human full-length NF-AT_(c) protein comprisingthe polypeptide sequence shown in FIG. 1, or polypeptides consistingessentially of a sequence shown in Table II.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example butnot limitation, in the human genome, the human CD4 gene is the cognategene to the mouse CD4 gene, since the sequences and structures of thesetwo genes indicate that they are highly homologous and both genes encodea protein which functions in signaling T cell activation through MHCclass II-restricted antigen recognition. Thus, the cognate murine geneto the human NF-AT_(c) gene is the murine gene which encodes anexpressed protein which has the greatest degree of sequence identity tothe human NF-AT_(c) protein and which exhibits an expression patternsimilar to that of the human NF-AT_(c) (e.g., expressed in T lineagecells). Preferred cognate NF-AT_(c) genes are: rat NF-AT_(c), rabbitNF-AT_(c), canine NF-AT_(c), nonhuman primate NF-AT_(c), porcineNF-AT_(c), bovine NF-AT_(c), and hamster NF-AT_(c).

The term “NF-AT_(c)-dependent gene” is used herein to refer to geneswhich: (1) have a NF-AT binding site (a site which can be specificallyfootprinted by NF-AT under suitable binding conditions) within about 10kilobases of the first coding sequence of said gene, and (2) manifest analtered rate of transcription, either increased or decreased, from amajor or minor transcriptional start site for said gene, wherein suchalteration in transcriptional rate correlates with the presence ofNF-AT_(c) polypeptide in NF-AT complexes, such as in an activated Tcell.

The term “altered ability to modulate” is used herein to refer to thecapacity to either enhance transcription or inhibit transcription of agene; such enhancement or inhibition may be contingent on the occurrenceof a specific event, such as T cell stimulation. This alteration will bemanifest as an inhibition of the transcriptional enhancement of the IL-2gene that normally ensues following T cell stimulation. The alteredability to modulate transcriptional enhancement or inhibition may affectthe inducible transcription of a gene, such as in the just-cited IL-2example, or may effect the basal level transcription of a gene, or both.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues. Agents are evaluated forpotential activity as immunomodulatory agents (e.g., immunosuppressants)by inclusion in screening assays described hereinbelow.

The term “candidate imunomodulatory agent” is used herein to refer to anagent which is identified by one or more screening method(s) of theinvention as a putative immuomodulatory agent. Some candidateimmunomodulatory agents may have therapeutic potential as drugs forhuman use.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes (e.g., ³H, ¹⁴C, 35S, ¹²⁵I, ¹³¹I), fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), biotinyl groups, predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

As used herein the terms “pathognomonic concentration”, “pathognomonicamount”, and “pathognomonic staining pattern” refer to a concentration,amount, or localization pattern, respectively, of a NF-AT_(c) protein ormRNA in a sample, that indicates the presence of a hypofunctional orhyperfunctional T cell condition or a predisposition to developing adisease, such as graft rejection. A pathognomonic amount is an amount ofa NF-AT_(c) protein or NF-AT_(c) mRNA in a cell or cellular sample thatfalls outside the range of normal clinical values that is established byprospective and/or retrospective statistical clinical studies.Generally, an individual having a neoplastic disease (e.g., lymphocyticleukemia) or T cell-mediated immune response will exhibit an amount ofNF-AT_(c) protein or mRNA in a cell or tissue sample that is higher thanthe range of concentrations that characterize normal, undiseasedindividuals; typically the pathognomonic concentration is at least aboutone standard deviation above the mean normal value, more usually it isat least about two standard deviations or more above the mean normalvalue. However, essentially all clinical diagnostic tests produce somepercentage of false positives and false negatives. The sensitivity andselectivity of the diagnostic assay must be sufficient to satisfy thediagnostic objective and any relevant regulatory requirements. Ingeneral, the diagnostic methods of the invention are used to identifyindividuals as disease candidates, providing an additional parameter ina differential diagnosis of disease made by a competent healthprofessional.

DETAILED DESCRIPTION

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, and nucleic acid chemistry andhybridization described below are those well known and commonly employedin the art. Standard techniques are used for recombinant nucleic acidmethods, polynucleotide synthesis, and microbial culture andtransformation (e.g., electroporation, lipofection). Generally enzymaticreactions and purification steps are performed according to themanufacturer's specifications. The techniques and procedures aregenerally performed according to conventional methods in the art andvarious general references (see, generally, Sambrook et al. MolecularCloning: A Laboratory Manual, 2d ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference) which are provided throughout this document. Theprocedures therein are believed to be well known in the art and areprovided for the convenience of the reader. All the informationcontained therein is incorporated herein by reference.

Oligonucleotides can be synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Methods for PCR amplification are described in the art (PCR Technology:Principles and Anolications for DNA Amplification ed. HA Erlich, FreemanPress, New York, N.Y. (1992); PCR Protocols: A Guide to Methods andApplications, eds. Innis, Gelfland, Snisky, and White, Academic Press,San Diego, Calif. (1990); Mattila et al. (1991) Nucleic Acids Res. 19:4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods andApplications 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; and U.S. Pat. No. 4,683,202, which are incorporated herein byreference).

Commonly assigned application U.S. Ser. No. 07/749,385 filed Aug. 22,1991 is incorporated herein by reference.

Cloning of NF-AT_(c) Polynucleotides

Genomic or cDNA clones encoding NF-AT_(c) may be isolated from clonelibraries (e.g., available from Clontech, Palo Alto, Calif.) usinghybridization probes designed on the basis of the nucleotide sequencesshown in FIG. 1 and using conventional hybridization screening methods(e.g., Benton W D and Davis R W (1977) Science 196: 180; Goodspeed etal. (1989) Gene 76: 1; Dunn et al. (1989) J. Biol. Chem. 264: 13057).Where a cDNA clone is desired, clone libraries containing cDNA derivedfrom T cell mRNA is preferred. Alternatively, synthetic polynucleotidesequences corresponding to all or part of the sequences shown in FIG. 1may be constructed by chemical synthesis of oligonucleotides.Additionally, polymerase chain reaction (PCR) using primers based on thesequence data disclosed in FIG. 1 may be used to amplify DNA fragmentsfrom genomic DNA, mRNA pools, or from cDNA clone libraries. U.S. Pat.Nos. 4,683,195 and 4,683,202 describe the PCR method. Additionally, PCRmethods employing one primer that is based on the sequence datadisclosed in FIG. 1 and a second primer that is not based on thatsequence data may be used. For example, a second primer that ishomologous to or complementary to a polyadenylation segment may be used.In an embodiment, a polynucleotide comprising the 2742 nucleotide-longsequence of FIG. 1 can be used. Alternative polynucleotides encoding the716 amino acid sequence of FIG. 1 can also be readily constructed bythose of skill in the art by using the degeneracy of the genetic code.Polynucleotides encoding amino acids 418 to 710 of the NF-AT_(c)sequence of FIG. 1 can also be constructed by those of skill in the art.

It is apparent to one of skill in the art that nucleotide substitutions,deletions, and additions may be incorporated into the polynucleotides ofthe invention. Nucleotide sequence variation may result from sequencepolymorphisms of various NF-AT_(c) alleles, minor sequencing errors, andthe like. However, such nucleotide substitutions, deletions, andadditions should not substantially disrupt the ability of thepolynucleotide to hybridize to one of the polynucleotide sequences shownin FIG. 1 under hybridization conditions that are sufficiently stringentto result in specific hybridization.

Specific hybridization is defined herein as the formation of hybridsbetween a probe polynucleotide (e.g., a polynucleotide of the inventionwhich may include substitutions, deletion, and/or additions) and aspecific target polynucleotide (e.g., a polynucleotide having thesequence in FIG. 1), wherein the probe preferentially hybridizes to thespecific target such that, for example, a single band corresponding toNF-AT_(c) mRNA (or bands corresponding to multiple alternative splicingproducts of the NF-AT_(c) gene) can be identified on a Northern blot ofRNA prepared from a suitable cell source (e.g., a T cell expressingNF-AT_(c)). Polynucleotides of the invention and recombinantly producedNF-AT_(c), and fragments or analogs thereof, may be prepared on thebasis of the sequence data provided in FIG. 1 according to methods knownin the art and described in Maniatis et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., (1989), Cold Spring Harbor, N.Y. and Bergerand Kimmel, Methods in Enzymology, Volume 152, Guide to MolecularCloning Techniques (1987), Academic Press, Inc., San Diego, Calif.,which are incorporated herein by reference.

NF-AT_(c) polynucleotides may be short oligonucleotides (e.g., 25-100bases long), such as for use as hybridization probes and PCR (or LCR)primers. NF-AT_(c) polynucleotide sequences may also comprise part of alarger polynucleotide (e.g., a cloning vector comprising a NF-AT_(c)clone) and may be fused, by polynucleotide linkage, in frame withanother polynucleotide sequence encoding a different protein (e.g.,glutathione S-transferase or β-galactosidase) for encoding expression ofa fusion protein. Typically, NF-AT_(c) polynucleotides comprise at least25 consecutive nucleotides which are substantially identical to anaturally-occurring NF-AT_(c) sequence (e.g., FIG. 1), more usuallyNF-AT_(c) polynucleotides comprise at least 50 to 100 consecutivenucleotides which are substantially identical to a naturally-occurringNF-AT_(c) sequence. However, it will be recognized by those of skillthat the minimum length of a NF-AT_(c) polynucleotide required forspecific hybridization to a NF-AT_(c) target sequence will depend onseveral factors: G/C content, positioning of mismatched bases (if any),degree of uniqueness of the sequence as compared to the population oftarget polynucleotides, and chemical nature of the polynucleotide (e.g.,methylphosphonate backbone, phosphorothiolate, etc.), among others.

For example but not limitation, suitable hybridization probes fordetecting and/or quantifying the presence of NF-AT_(c) mRNA in a samplegenerally comprise at least one, preferably at least two, and morepreferably all of the following human NF-AT_(c) sequences shown in TableI, or their complements:

TABLE I Selected Human NF-AT_(c) Polynucleotide Sequences 5′-TTC CTC CGGGGC GCG CGG CGT GAG CCC GGG GCG AGG-3′(SEQ ID NO:1); 5′-CAG CGC GGG GCGGCC ACT TCT CCT GTG CCT CCG CCC GCT GCT-3′(SEQ ID NO:2); 5′-GCC GCG CGGATG CCA AGC ACC AGC TTT CCA GTC CCT TCC AAG-3′(SEQ ID NO:3); 5′-CCA ACTTCA GCC CCG CCC TGC CGC TCC CCA CGG CGC ACT CCA-3′(SEQ ID NO:4); 5′-TTCAGA CCT CCA CAC CGG GCA TCA TCC CGC CGG CGG-3′(SEQ ID NO:5); 5′-GCC ACACCA GGC CTG ATG GGG CCC CTG CCC TGG AGA GTC CTC-3′(SEQ ID NO:6); 5′-AGTCTG CCC AGC CTG GAG GCC TAC AGA GAC CCC TCG TGC CTG-3′(SEQ ID NO:7);5′-GTG TCT CCC AAG ACC ACG GAC CCC GAG GAG GGC TTT CCC-3′(SEQ ID NO:8);5′-AGC TGG CTG GGT GCC CGC TCC TCC AGA CCC GCG TCC CCT TGC-3′(SEQ IDNO:9); 5′-TAC AGC CTC AAC GGC CGC CAG CCG CCC TAC TCA CCC CAC CAC-3′(SEQID NO:10); 5′-GAC CAC CGA CAG CAG CCT GGA CCT GGG AGA TGG CGT CCCTGT-3′(SEQ ID NO:11); 5′-CCT GGG CAG CCC CCC GCC CCC GGC CGA CTT CGC GCCCGA AGA-3′(SEQ ID NO:12); 5′-GCT CCC CTA CCA GTG GCG AAG CCC AAG CCC CTGTCC CCT ACG-3′(SEQ ID NO:13); 5′-CTT CGG ATT GAG GTG CAG CCC AAG TCC CACCAC CGA GCC CAC-3′(SEQ ID NO:14); 5′-CAT GGC TAC TTG GAG AAT GAG CCG CTGATG CTG CAG CTT TTC-3′(SEQ ID NO:15); 5′-AAG ACC GTG TCC ACC ACC AGC CACGAG GCT ATC CTC TCC AAC-3′(SEQ ID NO:16); 5′-TCA GCT CAG GAG CTG CCT CTGGTG GAG AAG CAG AGC ACG GAC-3′(SEQ ID NO:17); 5′-AAC GCC ATC TTT CTA ACCGTA AGC CGT GAA CAT GAG CGC G-3′(SEQ ID NO:18); 5′-AGA AAC GAC GTC GCCGTA AAG CAG CGT GGC GTG TGG CA-3′(SEQ ID NO:19); 5′-GCA TAC TCA GAT AGTCAC GGT TAT TTT GCT TCT TGC GAA TG-3′(SEQ ID NO:20).

Also for example but not limitation, the following pair of PCR primers(amplimers) may be used to amplify murine or human NF-AT_(c) sequences(e.g., by reverse transcriptase initiated PCR of RNA from NF-AT_(c)expressing cells):

(forward) 5′-AGGGCGCGGGCACCGGGGCGCGGGCAGGGCTCGGAG-3′(SEQ ID NO:21)(reverse) 5′-GCAAGAAGCAAAATAACCGTGACTATCTGAGTATGC-3′(SEQ ID NO:22)

If desired, PCR amplimers for amplifying substantially full-length cDNAcopies may be selected at the discretion of the practioner. Similarly,amplimers to amplify single NF-AT_(c) exons or portions of the NF-AT_(c)gene (murine or human) may be selected.

Each of these sequences may be used as hybridization probes or PCRamplimers to detect the presence of NF-AT_(c) mRNA, for example todiagnose a disease characterized by the presence of an elevatedNF-AT_(c) mRNA level in lymphocytes, or to perform tissue typing (i.e.,identify tissues characterized by the expression of NF-AT_(c) mRNA), andthe like. The sequences may also be used for detecting genomic NF-AT_(c)gene sequences in a DNA sample, such as for forensic DNA analysis (e.g.,by RFLP analysis, PCR product length(s) distribution, etc.) or fordiagnosis of diseases characterized by amplification and/orrearrangements of the NF-AT_(c) gene.

Production of NF-AT_(c) Polypeptides

The nucleotide and amino acid sequences shown in FIG. 1 enable those ofskill in the art to produce polypeptides corresponding to all or part ofthe full-length human NF-AT_(c) polypeptide sequence. Such polypeptidesmay be produced in prokaryotic or eukaryotic host cells by expression ofpolynucleotides encoding NF-AT_(c), or fragments and analogs thereof.Alternatively, such polypeptides may be synthesized by chemical methodsor produced by in vitro translation systems using a polynucleotidetemplate to direct translation. Methods for expression of heterologousproteins in recombinant hosts, chemical synthesis of polypeptides, andin vitro translation are well known in the art and are described furtherin Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2ndEd., Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods inEnzymology. Volume 152. Guide to Molecular Cloning Techniques (1987),Academic Press, Inc., San Diego, Calif.

Fragments or analogs of NF-AT_(c) may be prepared by those of skill inthe art. Preferred amino- and carboxy-termini of fragments or analogs ofNF-AT_(c) occur near boundaries of functional domains. For example, butnot for limitation, such functional domains include: (1) domainsconferring the property of binding to other NF-AT components (e.g.,AP-1), (2) domains conferring the property of nuclear localization instimulated T lymphocytes, and (3) domains conferring the property ofenhancing activation of T cells when expressed at sufficient levels insuch cells. Additionally, such functional domains might include: (1)domains conferring the property of binding to RNA polymerase species,(2) domains having the capacity to directly alter local chromatinstructure, which may comprise catalytic activities (e.g.,topoisomerases, endonucleases) and/or which may comprise structuralfeatures (e.g., zinc fingers, histone-binding moieties), and (3) domainswhich may interact with accessory proteins and/or transcription factors.

One method by which structural and functional domains may be identifiedis by comparison of the nucleotide and/or amino acid sequence data shownin FIG. 1 to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function, such as the zinc fingers. For example,the NAD-binding domains of dehydrogenases, particularly lactatedehydrogenase and malate dehydrogenase, are similar in conformation andhave amino acid sequences that are detectably homologous (Proteins,Structures and Molecular Principles, (1984) Creighton (ed.), W.H.Freeman and Company, New York, which is incorporated herein byreference). Further, a method to identify protein sequences that foldinto a known three-dimensional structure are known (Bowie et al. (1991)Science 253: 164). Thus, the foregoing examples demonstrate that thoseof skill in the art can recognize sequence motifs and structuralconformations that may be used to define structural and functionaldomains in the NF-AT_(c) sequences of the invention. One example of adomain is the rel similarity region from amino acid 418 to amino acid710 of the NF-AT_(c) polypeptide sequence of FIG. 1.

Additionally, computerized comparison of sequences shown in FIG. 1 toexisting sequence databases can identify sequence motifs and structuralconformations found in other proteins or coding sequences that indicatesimilar domains of the NF-AT_(c) protein. For example but not forlimitation, the programs GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package (Genetics Computer Group, 575Science Dr., Madison, Wis.) can be used to identify sequences indatabases, such as GenBank/EMBL, that have regions of homology with aNF-AT_(c) sequences. Such homologous regions are candidate structural orfunctional domains. Alternatively, other algorithms are provided foridentifying such domains from sequence data. Further, neural networkmethods, whether implemented in hardware or software, may be used to:(1) identify related protein sequences and nucleotide sequences, and (2)define structural or functional domains in NF-AT_(c) polypeptides(Brunak et al. (1991) J. Mol. Biol. 220: 49, which is incorporatedherein by reference).. For example, the 13-residue repeat motifs-SPRASVTEESWLG(SEQ ID NO:23)- and -SPRVSVTDDSWLG(SEQ ID NO:24)- areexamples of structurally related domains.

Fragments or analogs comprising substantially one or more functionaldomain may be fused to heterologous polypeptide sequences, wherein theresultant fusion protein exhibits the functional property(ies) conferredby the NF-AT_(c) fragment. Alternatively, NF-AT_(c) polypeptides whereinone or more functional domain have been deleted will exhibit a loss ofthe property normally conferred by the missing fragment.

By way of example and not limitation, the domain conferring the propertyof nuclear localization and/or interaction with AP1 may be fused toβ-galactosidase to produce a fusion protein that is localized to thenucleus and which can enzymatically convert a chromogenic substrate to achromophore.

Although one class of preferred embodiments are fragments having amino-and/or carboxy-termini corresponding to amino acid positions nearfunctional domains borders, alternative NF-AT_(c) fragments may beprepared. The choice of the amino- and carboxy-termini of such fragmentsrests with the discretion of the practitioner and will be made based onexperimental considerations such as ease of construction, stability toproteolysis, thermal stability, immunological reactivity, amino- orcarboxyl-terminal residue modification, or other considerations.

In addition to fragments, analogs of NF-AT_(c) can be made. Such analogsmay include one or more deletions or additions of amino acid sequence,either at the amino- or carboxy-termini, or internally, or both; analogsmay further include sequence transpositions. Analogs may also compriseamino acid substitutions, preferably conservative substitutions.Additionally, analogs may include heterologous sequences generallylinked at the amino- or carboxy-terminus, wherein the heterologoussequence(s) confer a functional property to the resultant analog whichis not indigenous to the native NF-AT_(c) protein. However, NF-AT_(c)analogs must comprise a segment of 25 amino acids that has substantialsimilarity to a portion of the amino acid sequence shown in FIG. 1,respectively, and which has at least one of the requisite functionalproperties enumerated in the Definitions (supra). Preferred amino acidsubstitutions are those which: (1) reduce susceptibility to proteolysis,(2) reduce susceptibility to oxidation, (3) alter post-translationalmodification of the analog, possibly including phosphorylation, and (4)confer or modify other physicochemical or functional properties of suchanalogs, possibly including interaction with calcineurin orphophorylation or dephosphorylation thereby. NF-AT_(c) analogs includevarious muteins of a NF-AT_(c) sequence other than thenaturally-occurring peptide sequence. For example, single or multipleamino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally-occurring NF-AT_(c) sequence(preferably in the portion of the polypeptide outside the functionaldomains).

Conservative amino acid substitution is a substitution of an amino acidby a replacement amino acid which has similar characteristics (e.g.,those with acidic properties: Asp and Glu). A conservative (orsynonymous) amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles, (1984) Creighton (ed.), W.H.Freeman and Company, New York; Introduction to Protein Structure,(1991), C. Branden and J. Tooze, Garland Publishing, New York, N.Y.; andThornton et al. (1991) Nature 354: 105; which are incorporated herein byreference).

Native NF-AT_(c) proteins, fragments thereof, or analogs thereof can beused as reagents in DNA binding assays and/or in vitro transcriptionassays for identifying agents that interfere with NF-AT function, saidagents are thereby identified as candidate drugs which may be used, forexample, to block T cell activation or treat T cell lymphocyticleukemias. Typically, in vitro DNA binding assays that measure bindingof NF-AT to DNA employ double-stranded DNA that contains an array of oneor more NF-AT recognition sites (as defined by specific footprinting ofnative NF-AT protein). The DNA is typically linked to a solid substrateby any of various means known to those of skill in the art; such linkagemay be noncovalent (e.g., binding to a highly charged surface such asNylon 66) or may be by covalent bonding (e.g., typically by chemicallinkage involving a nitrogen position in a nucleotide base, such asdiazotization). NF-AT_(c) polypeptides are typically labeled byincorporation of a radiolabeled amino acid. The labeled NF-AT_(c)polypeptide, usually reconstituted with an NF-AT nuclear component(e.g., AP-1 activity) to form an NF-AT complex, is contacted with theimmobilized DNA under aqueous conditions that permit specific binding incontrol binding reactions with a binding affinity of about 1×10⁶ M ⁻¹ orgreater (e.g., 10-250 mM NaCl or KCl and 5-100 mM Tris HCl pH 5-9,usually pH 6-8), generally including Zn⁺² and/or Mn⁺² and/or Mg⁺² in thenanomolar to micromolar range (1 nM to 999 μM). Specificity of bindingis typically established by adding unlabeled competitor at variousconcentrations selected at the discretion of the practitioner. Examplesof unlabeled protein competitors include, but are not limited to, thefollowing: unlabeled NF-AT_(c) polypeptide, bovine serum albumin, andnuclear protein extracts. Binding reactions wherein one or more agentsare added are performed in parallel with a control binding reaction thatdoes not include an agent. Agents which inhibit the specific binding ofNF-AT_(c) polypeptides to DNA, as compared to a control reaction, areidentified as candidate immunomodulatory drugs. Also, agents whichprevent transcriptional modulation by NF-AT in vitro are therebyidentified as candidate immunomodulatory drugs.

In addition to NF-AT_(c) polypeptides consisting only ofnaturally-occuring amino acids, NF-AT_(c) peptidomimetics are alsoprovided. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. DrugRes. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al.(1987) J. Med. Chem 30: 1229, which are incorporated herein byreference) and are usually developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biological or pharmacological activity), such as human NF-AT_(c),but have one or more peptide linkages optionally replaced by a linkageselected from the group consisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methodsknown in the art and further described in the following references:Spatola, A. F. in “Chemistry and Biochemistry of Amino Acids, Peptides,and Proteins,” B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983); Soatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,“Peptide Backbone Modifications” (general review); Morley, J. S., TrendsPharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., Int JPent Prot Res (1979) 14:177-185 (—CH₂NH—, CH₂CH₂—); Spatola, A. F. etal., Life Sci (1986) 33:1243-1249 (—CH₂—S); Hann, M. M., J Chem SocPerkin Trans I (1982) 307-314 (—CH—CH—, cis and trans); Almquist, R. G.et al., J Med Chem (1980) 23:1392-1398 (—COCH₂—); Jennings-White, C. etal., Tetrahedron Lett (1982) 23:2533 (—COCH₂—); Szelke, M. et al.,European Appln. EP 45665 (1982) CA: 97:39405 (1982) (—CH(OH)CH₂—);Holladay, M. W. et al., Tetrahedron Lett (1983) 24:4401-4404(—C(OH)CH₂—); and Hruby, V. J., Life Sci (1982) 31:189-199 (—CH₂—S—);each of which is incorporated herein by reference. A particularlypreferred non-peptide linkage is —CH₂NH—. Such peptide mimetics may havesignificant advantages over polypeptide embodiments, including, forexample: more economical production, greater chemical stability,enhanced pharmacological properties (half-life, absorption, potency,efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., immunoglobulinsuperfamily molecules) to which the peptidomimetic binds to produce thetherapeutic effect. Derivitization (e.g., labelling) of peptidomimeticsshould not substantially interfere with the desired biological orpharmacological activity of the peptidomimetic. Peptidomimetics ofNF-AT_(c) may be used as competitive or noncompetitive agonists orantagonists of NF-AT_(c) function. For example, a NF-AT_(c)peptidomimetic administered to a stimulated T cell containing NF-AT_(c)and may compete with the naturally-occurring NF-AT_(c) and reduce NF-ATactivity. Alternatively, an NF-AT_(c) peptidomimetic administerd to a Tcell lacking NF-AT_(c) may induce T cell activation or the like.

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides (including cyclized peptides) comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch (1992) Ann. Rev. Biochem. 61: 387, incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

The amino acid sequences of NF-AT_(c) polypeptides identified hereinwill enable those of skill in the art to produce polypeptidescorresponding to NF-AT_(c) peptide sequences and sequence variantsthereof. Such polypeptides may be produced in prokaryotic or eukaryotichost cells by expression of polynucleotides encoding a NF-AT_(c) peptidesequence, frequently as part of a larger polypeptide. Alternatively,such peptides may be synthesized by chemical methods. Methods forexpression of heterologous proteins in recombinant hosts, chemicalsynthesis of polypeptides, and in vitro translation are well known inthe art and are described further in Maniatis et al., Molecular Cloning:A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Bergerand Kimmel, Methods in Enzymology, Volume 152. Guide to MolecularCloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;Merrifield, J. (1969) J. Am. Chem. Soc. 91: 501; Chaiken I. M. (1981)CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989) Science 243: 187;Merrifield, B. (1986) Science 232: 342; Kent, S. B. H. (1988) Ann. Rev.Biochem. 57: 957; and Offord, R. E. (1980) Semisynthetic Proteins, WileyPublishing, which are incorporated herein by reference).

Production and Applications of α-NF-AT_(c) Antibodies

Native NF-AT_(c) proteins, fragments thereof, or analogs thereof, may beused to immunize an animal for the production of specific antibodies.These antibodies may comprise a polyclonal antiserum or may comprise amonoclonal antibody produced by hybridoma cells. For general methods toprepare antibodies, see Antibodies: A Laboratory Manual, (1988) E.Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., which is incorporated herein by reference.

For example but not for limitation, a recombinantly produced fragment ofhuman NF-AT_(c) can be injected into a rat along with an adjuvantfollowing immunization protocols known to those of skill in the art soas to generate an immune response. Typically, approximately at least1-50 μg of a NF-AT_(c) fragment or analog is used for the initialimmunization, depending upon the length of the polypeptide.Alternatively or in combination with a recombinantly produced NF-AT_(c)polypeptide, a chemically synthesized peptide having a NF-AT_(c)sequence (e.g., peptides exemplified in Table II, infra) may be used asan immunogen to raise antibodies which bind a NF-AT_(c) protein, such asthe native human NF-AT_(c) polypeptide having the sequence shownessentially in FIG. 1 or the native human NF-AT_(c) polypeptide isoform.Immunoglobulins which bind the recombinant fragment with a bindingaffinity of at least 1×10⁷ M⁻¹ can be harvested from the immunizedanimal as an antiserum, and may be further purified byimmunoaffinitychromatography or other means. Additionally, spleen cells are harvestedfrom the immunized animal (typically rat or mouse) and fused tomyelomacells to produce a bank of antibody-secreting hybridoma cells. The bankof hybridomas can be screened for clones that secrete immunoglobulinswhich bind the recombinantly produced NF-AT_(c) polypeptide (orchemically synthesized NF-AT_(c) polypeptide) with an affinity of atleast 1×10⁶ M⁻¹. Animals other than mice and rats may be used to raiseantibodies; for example, goats, rabbits, sheep, and chickens may also beemployed to raise antibodies reactive with a NF-AT_(c) protein.Transgenic mice having the capacity to produce substantially humanantibodies also may be immunized and used for a source of α-NF-AT_(c)antiserum and/or for making monoclonal-secreting hybridomas.

Bacteriophage antibody display libraries may also be screened forbinding to a NF-AT_(c) polypeptide, such as a full-length humanNF-AT_(c) protein, a NF-AT_(c) fragment (e.g., a peptide having asequence shown in Table II, infra), or a fusion protein comprising aNF-AT_(c) polypeptide sequence of at least 14 contiguous amino acids asshown in FIG. 1 or a polypeptide sequence of Table II (infra).Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which may be screened asbacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:2432). Various embodiments of bacteriophage antibody display librariesand lambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991)Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147:3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol.Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992)Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007;Lowman,et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science258: 1313, incorporated herein by reference). Typically, abacteriophagelantibody display library is screened with a NF-AT_(c)polypeptide that is immobilized (e.g., by covalent linkage to achromatography resin to enrich for reactive phage by affinitychromatography) and/or labeled (e.g., to'screen plaque or colony lifts).

NF-AT_(c) polypeptides which are useful as immunogens, for diagnosticdetection of α-NF-AT_(c) antibodies in a sample, for diagnosic detectionand quantitation of NF-AT_(c) protein in a sample (e.g., by standardizedcompetitive ELISA), or for screening a bacteriophage antibody displaylibrary, are suitably obtained in substantially pure form, that is,typically about 50 percent (w/w) or more purity, substantially free ofinterfering proteins and contaminants. Preferably, these polypeptidesare isolated or synthesized in a purity of at least 80 percent (w/w)and, more preferably, in at least about 95 percent (w/w) purity, beingsubstantially free of other proteins of humans, mice, or othercontaminants. Preferred immunogens comprise at least one NF-AT_(c)polypeptide sequence shown in Table II, either as a discrete peptide oras part of a fusion polypeptide (e.g., with a β-galactosidase orglutathione S-transferase sequence). NF-AT_(c) immunogens comprise atleast one, typically several of such immunogenic epitopes.

For some applications of these antibodies, such as identifyingimmunocrossreactive proteins, the desired antiserum or monoclonalantibody(ies) is/are not monospecific. In these instances, it may bepreferable to use a synthetic or recombinant fragment of NF-AT_(c) as anantigen rather than using the entire native protein. More specifically,where the object is to identify immunocrossreactive polypeptides thatcomprise a particular structural moiety, such as a DNA-binding domain,it is preferable to use as an antigen a fragment corresponding to partor all of a commensurate structural domain in the NF-AT_(c) protein.Production of recombinant or synthetic fragments having such definedamino- and carboxy-termini is provided by the NF-AT_(c) sequences shownin FIG. 1.

If an antiserum is raised to a NF-AT_(c) fusion polypeptide, such as afusion protein comprising a NF-AT_(c) immunogenic epitope fused toβ-galactosidase or glutathione S-transferase, the antiserum ispreferably preadsorbed with the non-NF-AT_(c) fusion partner (e.g,β-galactosidase or glutathione S-transferase) to deplete the antiserumof antibodies that react (i.e., specifically bind to) the non-NF-AT_(c)portion of the fusion protein that serves as the immunogen. Monoclonalor polyclonal antibodies which bind to the human and/or murine NF-AT_(c)protein can be used to detect the presence of human or murine NF-AT_(c)polypeptides in a sample, such as a Western blot of denatured protein(e.g., a nitrocellulose blot of an SDS-PAGE) obtained from a lymphocytesample of a patient. Preferably quantitative detection is performed,such as by denistometric scanning and signal integration of a Westernblot. The monoclonal or polyclonal antibodies will bind to the denaturedNF-AT_(c) epitopes and may be identified visually or by other opticalmeans with a labeled second antibody, or labeled Staphylococcus aureusprotein A by methods known in the art. Frequently, denatured NF-AT_(c)will be used as the target antigen so that more epitopes may beavailable for binding.

TABLE II Selected Human NF-AT_(c) Antigen Peptides -NAIFLTVSREHERVGC(SEQID NO:25)-; -AKTDRDLCKPNSLVVEIPPFRN(SEQ ID NO:31)-;-LHGYLENEPLMLQLFIGT(SEQ ID NO:26)-; -EVQPKSHHRAHYETEGSR-(SEQ ID NO:32);-PSTSPRASVTEESWLG(SEQ ID NO:27)-; -SPRVSVTDDSWLGNT-(SEQ ID NO:33);-GPAPRAGGTMKSAEEEHYG(SEQ ID NO:28)-; -SHHRAHYETEGSRGAV-(SEQ ID NO:34);-ASAGGHPIVQ-(SEQ ID NO:29); -LRNSDIELRKGETDIGR-(SEQ ID NO:35);-NTRVRLVFRV-(SEQ ID NO:30); -TLSLQVASNPIEC-(SEQ ID NO:36).

Such NF-AT_(c) sequences as shown in Tables II may be used as animmunogenic peptide directly (e.g., to screen bacteriophage antibodydisplay libraries or to immunize a rabbit), or may be conjugated to acarrier macromolecule (e.g., BSA) or may compose part of a fusionprotein to be used as an immunogen. A preferred NF-AT_(c) polypeptidecomprises the following amino acids sequences:

-PSTSPRASVTEESWLG-;(SEQ ID NO:32) SPRVSVTDDSWLGNT-;(SEQ ID NO:33)-SHHRAHYETEGSRGAV-;(SEQ ID NO:34) NAIFLTVSREHERVGC-;(SEQ ID NO:35)

and may comprise other intervening and/or terminal sequences; generallysuch polypeptides are less than 1000 amino acids in length, more usuallyless than about 500 amino acids in length; often spacer peptidesequences or terminal peptide sequences, if present, correspond tonaturally occurring polypeptide sequences, generally mammalianpolypeptide sequences. One application of the preferred NF-AT_(c)polypeptide just recited is as a commercial immunogen to raiseα-NF-AT_(c) antibodies in a suitable animal and/or as a commercialimmunodiagnostic reagent for quantitative ELISA (e.g., competitiveELISA) or competitive RIA in conjunction with the anti-NF-AT_(c)antibodies provided by the invention, such as for calibration ofstandardization of such immunoassays for staging or diagnosis ofNF-AT_(c)-expressing lymphocytic leukemias in humans or cell typing oridentification of T cells (such as activated T cells and/or activatableT cells). The preferred NF-AT_(c) polypeptide just recited will findmany other uses in addition to serving as an immunogen or immunologicalreagent. One or more of the above-listed sequences may be incorporatedinto a fusion protein with fusion partner such as human serum albumin,GST, etc. For such fusion proteins in excess of 1000 amino acids,deletions in the fusion partner (albumin) moiety may be made to bringthe size to about 1000 amino acids or less, if desired.

In some embodiments, it will be desirable to employ a polyvalentNF-AT_(c) antigen, comprising at least two NF-AT_(c) immunogenicepitopes in covalent linkage, usually in peptide linkage. Suchpolyvalent NF-AT_(c) antigens typically comprise multiple NF-AT_(c)antigenic peptides from the same species (e.g., human or mouse), but maycomprise a mix of antigenic peptides from NF-AT_(c) proteins ofdifferent species (i.e., an interspecies NF-AT_(c) polyvalent antigen).Frequently, the spatial order of the antigenic peptide sequences in theprimary amino acid sequence of a polyvalent antigen occurs in the sameorientation as in the naturally occurring NF-AT_(c) protein (i.e., afirst antigenic peptide sequence that is amino-terminal to a secondantigenic peptide sequence in a naturally occurring NF-AT_(c)r proteinwill be amino-terminal to said second antigenic peptide sequence in apolyvalent antigen. Frequently, spacer peptide sequences will be used tolink antigenic peptide sequences in a polyvalent antigen, such spacerpeptide sequences may be predetermined, random, or psuedorandomsequences. Spacer peptide sequences may correspond to sequences known tobe non-immunogenic to the animal which is to be immunized with thepolyvalent antigen, such as a sequence to which the animal has beentolerized. Although many examples of such polyvalent antigens may begiven, the following embodiment is provided for illustration and notlimitation:

-NAIFLTVSREHERVGC(SEQ ID NO:25)-(aa1) -AKTDRDLCKPNSLVVEIPPFRN(SEQ IDNO:31)-(aa2)- VKASAGGHPIVQL(SEQ ID NO:37)

where (aa1) and (aa2) are peptide spacers of at least one amino acid andless than 1000 amino acids; aa1 is a peptide sequence selectedindependently from the aa2 peptide sequence; the length of aa1 (whichmay be composed of multiple different amino acids) is independent of thelength of aa2 (which may be composed of multiple different amino acids).

Immunogenic NF-AT_(c) peptides may be used to immunize an animal toraise anti-NF-AT_(c) antibodies and/or as a source of spleen cells formaking a hybridoma library from which to select hybridoma clones whichsecrete a monoclonal antibody which binds to a NF-AT_(c) protein with anaffinity of 1×10⁷ M⁻¹ or greater, preferably at least 1×10⁸ M⁻¹ to 1×10⁹M⁻¹. Such immunogenic NF-AT_(c) peptides can also be used to screenbacteriophage antibody display libraries directly.

One use of such antibodies is to screen cDNA expression libraries,preferably containing cDNA derived from human or murine mRNA fromvarious tissues, for identifying clones containing cDNA inserts whichencode structurally-related, immunocrossreactive proteins, that arecandidate novel transcription factors or chromatin proteins. Suchscreening of cDNA expression libraries is well known in the art, and isfurther described in Young et al., Proc. Natl. Acad. Sci. U.S.A.80:1194-1198 (1983), which is incorporated herein by reference] as wellas other published sources. Another use of such antibodies is toidentify and/or purify immunocrossreactive proteins that arestructurally or evolutionarily related to the native NF-AT_(c) proteinor to the corresponding NF-AT_(c) fragment (e.g., functional domain;DNA-binding domain) used to generate the antibody. It is believed thatsuch antibodies will find commercial use as such reagents for researchapplications, just as other antibodies (and biological reagents—such asrestriction enzymes and polymerases) are sold commercially.

Various other uses of such antibodies are to diagnose and/or stageleukemias or other immunological disease states, and for therapeuticapplication (e.g., as cationized antibodies or by targeted liposomaldelivery) to treat neoplasia, hyperimmune function, graft rejection, andthe like.

An example of an NF-AT_(c) polypeptide is a polypeptide having thesequence:

MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASSNVSPALPLPTAHS (SEQ IDNO:38) TLPAPCHNLQTSTPGIIPPADHPSGYGAALDGCPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKRSPSTATLSLPSLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASPQTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPQHSPSTSPRASVTEESWLGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTLEQPPSVALKVEPVGEDLGSPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPKPLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCACILKLRNSDIELRKGETDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANGNAIFLTVSREHERVGCFF

NF-AT_(c) Polynucleotides

Disclosure of the full coding sequence for human NF-AT_(c) shown in FIG.1 makes possible the construction of isolated polynucleotides that candirect the expression of NF-AT_(c), fragments thereof, or analogsthereof. Further, the sequences in FIG. 1 make possible the constructionof nucleic acid hybridization probes and PCR primers that can be used todetect RNA and DNA sequences encoding NF-AT_(c).

Polynucleotides encoding full-length NF-AT_(c) or fragments or analogsthereof, may include sequences that facilitate transcription (expressionsequences) and translation of the coding sequences, such that theencoded polypeptide product is produced. Construction of suchpolynucleotides is well known in the art and is described further inManiatis et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989),Cold Spring Harbor, N.Y. For example, but not for limitation, suchpolynucleotides can include a promoter, a transcription termination site(polyadenylation site in eukaryotic expression hosts), a ribosomebinding site, and, optionally, an enhancer for use in eukaryoticexpression hosts, and, optionally, sequences necessary for replicationof a vector. A typical eukaryotic expression cassette will include apolynucleotide sequence encoding a NF-AT_(c) polypeptide linkeddownstream (i.e., in translational reading frame orientation;polynucleotide linkage) of a promoter such as the HSV tk promoter or thepgk (phosphoglycerate kinase) promoter, optionally linked to an enhancerand a downstream polyadenylation site (e.g., an SV40 large T Ag poly Aaddition site).

A preferred NF-AT_(c) polynucleotide encodes a NF-AT_(c) polypeptidethat comprises at least one of the following amino acids sequences:

-NAIFLTVSREHERVGC-(SEQ ID NO:25); -AKTDRDLCKPNSLVVEIPPFRN-(SEQ IDNO:31); -LHGYLENEPLMLQLFIGT-(SEQ ID NO:26); -EVQPKSHHRAHYETEGSR-(SEQ IDNO:32); -PSTSPRASVTEESWLG-(SEQ ID NO:27); -SPRVSVTDDSWLGNT-(SEQ IDNO:33); -GPAPRAGGTMKSAEEEHYG-(SEQ ID NO:28); -SHHRAHYETEGSRGAV-(SEQ IDNO:34); -ASAGGHPIVQ-(SEQ ID NO:29); -LRNSDIELRKGETDIGR-(SEQ ID NO;35);-NTRVRLVFRV-(SEQ ID NO:30); -TLSLQVASNPIEC-(SEQ ID NO:36).

The degeneracy of the genetic code gives a finite set of polynucleotidesequences encoding these amino acid sequences; this set of degeneratesequences may be readily generated by hand or by computer usingcommercially available software (Wisconsin Genetics Software PackageRelease 7.0). Thus, isolated polynucleotides typically less thanapproximately 10,000 nucleotides in length and comprising sequencesencoding each of the following amino acid sequences:

-NAIFLTVSREHERVGC-(SEQ ID NO:25); -AKTDRDLCKPNSLVVEIPPFRN-(SEQ IDNO:31); -LHGYLENEPLMLQLFIGT-(SEQ ID NO:26); -EVQPKSHHRAHYETEGSR-(SEQ IDNO:32); -PSTSPRASVTEESWLG-(SEQ ID NO:27); -SPRVSVTDDSWLGNT-(SEQ IDNO:33); -GPAPRAGGTMKSAEEEHYG-(SEQ ID NO:28); -SHHRAHYETEGSRGAV-(SEQ IDNO:34); -ASAGGHPIVQ-(SEQ ID NO:29); -LRNSDIELRKGETDIGR-(SEQ ID NO;35);-NTRVRLVFRV-(SEQ ID NO:30); -TLSLQVASNPIEC-(SEQ ID NO:36).

are provided and may be used for, among other uses, the expression of aNF-AT_(c) polypeptide which can be used as an immunogen, immunologicalreagent, and the like. Such polynucleotides typically comprise anoperably linked promoter for driving expression in a suitableprokaryotic or eukaryotic host cell. One exemplification of such apolynucleotide is the human NF-AT_(c) cDNA sequence of FIG. 1 cloned inoperable linkage to the mammalian expression vector pSRα, manyalternative embodiments will be apparent to those of skill in the art,including the use of alternative expression vectors (e.g., pBC12BI andp91023(B); Hanahan J (1983) J. Mol. Biol. 166: 577; Cullen et al. (1985)J. Virol. 53: 515; Lomedico PT (1982) Proc. Natl. Acad. Sci. (U.S.A.)79: 5798; Morinaga et al. (1984) Bio/Technology 2: 636).

Additionally, where expression of a polypeptide is not desired,polynucleotides of this invention need not encode a functional protein.Polynucleotides of this invention may serve as hybridization probesand/or PCR primers (amplimers) and/or LCR oligomers for detectingNF-AT_(c) RNA or DNA sequences.

Alternatively, polynucleotides of this invention may serve ashybridization probes or primers for detecting RNA or DNA sequences ofrelated genes, such genes may encode structurally or evolutionarilyrelated proteins. For such hybridization and PCR applications, thepolynucleotides of the invention need not encode a functionalpolypeptide. Thus, polynucleotides of the invention may containsubstantial deletions, additions, nucleotide substitutions and/ortranspositions, so long as specific hybridization or specificamplification to the NF-AT_(c) sequence is retained.

Specific hybridization is defined hereinbefore, and can be roughlysummarized as the formation of hybrids between a polynucleotide of theinvention (which may include substitutions, deletions, and/or additions)and a specific target polynucleotide such as human NF-AT_(c) mRNA sothat a single band is identified corresponding to each NF-AT_(c) isoformon a Northern blot of RNA prepared from T cells (i.e., hybridization andwashing conditions can be established that permit detection of discreteNF-AT_(c) mRNA band(s)). Thus, those of ordinary skill in the art canprepare polynucleotides of the invention, which may include substantialadditions, deletions, substitutions, or transpositions of nucleotidesequence as compared to sequences shown in FIG. 1 and determine whetherspecific hybridization is a property of the polynucleotide by performinga Northern blot using RNA prepared from a T lymphocyte cell line whichexpresses NF-AT_(c) mRNA and/or by hybridization to a NF-AT_(c) DNAclone (cDNA or genomic clone).

Specific amplification is defined as the ability of a set of PCRamplimers, when used together in a PCR reaction with a NF-AT_(c)polynucleotide, to produce substantially a single major amplificationproduct which corresponds to a NF-AT_(c) gene sequence or mRNA sequence.Generally, human genomic DNA or mRNA from NF-AT_(c) expressing humancells (e.g., Jurkat cell line) is used as the template DNA sample forthe PCR reaction. PCR amplimers that exhibit specific amplification aresuitable for quantitative determination of NF-AT_(c) mRNA byquantitative PCR amplification. NF-AT_(c) allele-specific amplificationproducts, although having sequence and/or length polymorphisms, areconsidered to constitute a single amplification product for purposes ofthis definition.

Generally, hybridization probes comprise approximately at least 25consecutive nucleotides of a sequence shown in FIG. 1 (for human andmurine NF-AT_(c) detection, respectively), preferably the hybridizationprobes contain at least 50 consecutive nucleotides of a sequence shownin FIG. 1, and more preferably comprise at least 100 consecutivenucleotides of a sequence shown in FIG. 1. PCR amplimers typicallycomprise approximately 25 to 50 consecutive nucleotides of a sequenceshown in FIG. 1, and usually consist essentially of approximately 25 to50 consecutive nucleotides of a sequence shown in FIG. 1 with additionalnucleotides, if present, generally being at the 5′ end so as not tointerfere with polymerase-mediated chain extension. PCR amplimer designand hybridization probe selection are well within the scope ofdiscretion of practioners of ordinary skill in the art.

Methods Relating to Genetic Disease

In one preferred embodiment of the invention, hybridization probes thatspecifically identify the NF-AT_(c) gene may be used in methods fordiagnosing genetic disease. For example, but not for limitation, thegenetic disease thus diagnosed may involve a lesion in the relevantNF-AT_(c) structural or regulatory sequences, or may involve a lesion ina genetic locus closely linked to the NF-AT_(c) locus and which can beidentified by restriction fragment length polymorphism or DNA sequencepolymorphism at the linked NF-AT_(c) locus. In a further preferredembodiment, NF-AT_(c) gene probes are used to diagnose or identifygenetic disease involving predisposition to immunological disease,wherein the amount or functionality of endogenous NF-AT_(c) issufficient for the individual to exhibit an increased probability ofdeveloping an immune disease, particularly an immune deficiency,arthritis, or autoimmune disease.

Antisense Polynucleotides

Additional embodiments directed to modulation of T cell activationinclude methods that employ specific antisense polynucleotidescomplementary to all or part of the sequences shown in FIG. 1. Suchcomplementary antisense polynucleotides may include nucleotidesubstitutions, additions, deletions, or transpositions, so long asspecific hybridization to the relevant target sequence corresponding toFIG. 1 is retained as a functional property of the polynucleotide.Complementary antisense polynucleotides include soluble antisense RNA orDNA oligonucleotides which can hybridize specifically to NF-AT_(c) mRNAspecies and prevent transcription of the mRNA species and/or translationof the encoded polypeptide (Ching et al. (1989) Proc. Natl. Acad. Sci.U.S.A. 86: 10006; Broder et al. (1990) Ann. Int. Med. 113: 604; Loreauet al. (1990) FEBS Letters 274: 53; Holcenberg et al., WO91/11535; U.S.Ser. No. 07/530,165; WO91/09865; WO91/04753; WO90/13641; and EP 386563,each of which is incorporated herein by reference). The antisensepolynucleotides therefore inhibit production of NF-AT_(c) polypeptides.Since NF-AT_(c) protein expression is associated with T lymphocyteactivation, antisense polynucleotides that prevent transcription and/ortranslation of mRNA corresponding to NF-AT_(c) polypeptides may inhibitT cell activation and/or reverse the the activated phenotype of T cells.Compositions containing a therapeutically effective dosage of NF-AT_(c)antisense polynucleotides may be administered for treatment of immunediseases, including lymphocytic leukemias, and for inhibition oftransplant rejection reactions, if desired. Antisense polynucleotides ofvarious lengths may be produced, although such antisense polynucleotidestypically comprise a sequence of about at least 25 consecutivenucleotides which are substantially identical to a naturally-occurringNF-AT_(c) polynucleotide sequence, and typically which are identical-toa sequence shown in FIG. 1.

Antisense polynucleotides may be produced from a heterologous expressioncassette in a transfectant cell or transgenic cell, such as a transgenicpluripotent hematopoietic stem cell used to reconstitute-all or part ofthe hematopoietic stem cell population of an individual. Alternatively,the antisense polynucleotides may comprise soluble oligonucleotides thatare administered to the external milieu, either in the culture medium invitro or in the circulatory system or interstitial fluid in vivo.Soluble antisense polynucleotides present in the external milieu havebeen shown to gain access to the cytoplasm and inhibit translation ofspecific mRNA species. In some embodiments the antisense polynucleotidescomprise methylphosphonate moieties. For general methods relating toantisense polynucleotides, see Antisense RNA and DNA, (1988), D. A.Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Isolation of the Cognate Human NF-AT_(c) Gene

The human homolog of the NF-AT_(c) cDNA is identified and isolated byscreening a human genomic clone library, such as a human genomic libraryin yeast artificial chromosomes, cosmids, or bacteriophage λ (e.g., λCharon 35), with a polynucleotide probe comprising a sequence of aboutat least 24 contiguous nucleotides (or their complement) of the cDNAsequence shown in FIG. 1. Typically, hybridization and washingconditions are performed at high stringency according to conventionalhybridization procedures. Positive clones are isolated and sequenced.For illustration and not for limitation, a full-length polynucleotidecorresponding to the sequence of FIG. 1 may be labeled and used as ahybridization probe to isolate genomic clones from a human or murinegenomic clone library in λEMBL4 or λGEM11 (Promega Corporation, Madison,Wis.); typical hybridization conditions for screening plaque lifts(Benton and Davis (1978) Science 196:180) can be: 50% formamide, 5×SSCor SSPE, 1-5×Denhardt's solution, 0.1-1% SDS, 100-200 μg shearedheterologous DNA or tRNA, 0-10% dextran sulfate, 1×10⁵ to 1×10⁷ cpm/mlof denatured probe with a specific activity of about 1×10⁸ cpm/μg, andincubation at 42° C. for about 6-36 hours. Prehybridization conditionsare essentially identical except that probe is not included andincubation time is typically reduced. Washing conditions are typically1-3×SSC, 0.1-1% SDS, 50-70° C. with change of wash solution at about5-30 minutes.

Nonhuman NF-AT_(c) cDNAs and genomic clones (i.e., cognate nonhumanNF-AT_(c) genes) can be analogously isolated from various nonhuman cDNAand genomic clone libraries available in the art (e.g., Clontech, PaloAlto, Calif.) by using probes based on the sequences shown in FIG. 1,with hybridization and washing conditions typically being less stringentthan for isolation of human NF-AT_(c) clones.

Polynucleotides comprising sequences of approximately at least 30-50nucleotides, preferably at least 100 nucleotides, corresponding to orcomplementary to the nucleotide sequences shown in FIG. 1 can serve asPCR primers and/or hybridization probes for identifying and isolatinggermline genes corresponding to NF-AT_(c). These germline genes may behuman or may be from a related mammalian species, preferably rodents orprimates. Such germline genes may be isolated by various methodsconventional in the art, including, but not limited to, by hybridizationscreening of genomic libraries in bacteriophage λ or cosmid libraries,or by PCR amplification of genomic sequences using primers derived fromthe sequences shown in FIG. 1. Human genomic libraries are publiclyavailable or may be constructed de novo from human DNA.

Genomic clones of NF-AT_(c), particularly of the murine cognateNF-AT_(c) gene, may be used to construct homologous targeting constructsfor generating cells and transgenic nonhuman animals having at least onefunctionally disrupted NF-AT_(c) allele, preferably homozygous forknocked out NF-AT_(c) alleles. Guidance for construction of homologoustargeting constructs may be found in the art, including: Rahemtulla etal. (1991) Nature 353: 180; Jasin et al. (1990) Genes Devel. 4: 157; Kohet al. (1992) Science 256: 1210; Molina et al. (1992) Nature 357: 161;Grusby et al. (1991) Science 253: 1417; Bradley et al. (1992)Bio/Technology 10: 534, incorporated herein by reference). Homologoustargeting can be used to generate so-called “knockout” mice, which areheterozygous or homozygous for an inactivated NF-AT_(c) allele. Suchmice may be sold commercially as research animals for investigation ofimmune system development, neoplasia, T cell activation, signaltransduction, drug sreening, and other uses.

Chimeric targeted mice are derived according to Hogan, et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed., IRL Press, Washington, D.C.,(1987) which are incorporated herein by reference. Embryonic stem cellsare manipulated according to published procedures (Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRLPress, Washington, D.C. (1987); Zjilstra et al. (1989) Nature 342:435;and Schwartzberg et al. (1989) Science 246: 799, each of which isincorporated herein by reference).

Additionally, a NF-AT_(c) cDNA or genomic gene copy may be used toconstruct transgenes for expressing NF-AT_(c) polypeptides at highlevels and/or under the transcriptional control of transcription controlsequences which do not naturally occur adjacent to the NF-AT_(c) gene.For example but not limitation, a constitutive promoter (e.g., a HSV-tkor pgk promoter) or a cell-lineage specific transcriptional regulatorysequence (e.g., a CD4 or CD8 gene promoter/enhancer) may be operablylinked to a NF-AT_(c)-encoding polynucleotide sequence to form atransgene (typically in combination with a selectable marker such as aneo gene expression cassette). Such transgenes can be introduced intocells (e.g., ES cells, hematopoietic stem cells) and transgenic cellsand transgenic nonhuman animals may be obtained according toconventional methods. Transgenic cells and/or transgenic nonhumananimals may be used to screen for antineoplastic agents and/or to screenfor potential immunomodulatory agents, as overexpression of NF-AT_(c) orinappropriate expression of NF-AT_(c) may result in a hyperimmune stateor enhance graft rejection reactions.

Identification and Isolation of Proteins that Bind NF-AT_(c)

Proteins that bind to NF-AT_(c) and/or a NFAT-DNA complex arepotentially important transcriptional regulatory proteins. Such proteinsmay be targets for novel immunomodulatory agents. These proteins arereferred to herein as accessory proteins. Accessory proteins may beisolated by various methods known in the art.

One preferred method of isolating accessory proteins is by contacting aNF-AT_(c) polypeptide to an antibody that binds the NF-AT_(c)polypeptide, and isolating resultant immune complexes. These immunecomplexes may contain accessory proteins bound to the NF-AT_(c)polypeptide. The accessory proteins may be identified and isolated bydenaturing the immune complexes with a denaturing agent and, preferably,a reducing agent. The denatured, and preferably reduced, proteins can beelectrophoresed on a polyacrylamide gel. Putative accessory proteins canbe identified on the polyacrylamide gel by one or more of various wellknown methods (e.g., Coomassie staining, Western blotting, silverstaining, etc.), and isolated by resection of a portion of thepolyacrylamide gel containing the relevant identified polypeptide andelution of the polypeptide from the gel portion.

A putative accessory protein may be identified as an accessory proteinby demonstration that the protein binds to NF-AT_(c) and/or a NFAT-DNAcomplex. Such binding may be shown in vitro by various means, including,but not limited to, binding assays employing a putative accessoryprotein that has been renatured subsequent to isolation by apolyacrylamide gel electrophoresis method. Alternatively, binding assaysemploying recombinant or chemically synthesized putative accessoryprotein may be used. For example, a putative accessory protein may beisolated and all or part of its amino acid sequence determined bychemical sequencing, such as Edman degradation. The amino acid sequenceinformation may be used to chemically synthesize the putative accessoryprotein. The amino acid sequence may also be used to produce arecombinant putative accessory protein by: (1) isolating a cDNA cloneencoding the putative accessory protein by screening a cDNA library withdegenerate oligonucleotide probes according to the amino acid sequencedata, (2) expressing the cDNA in a host cell, and (3) isolating theputative accessory protein. Alternatively, a polynucleotide encoding aNF-AT_(c) polypeptide may be constructed by oligonucleotide synthesis,placed in an expression vector, and expressed in a host cell.

Putative accessory proteins that bind NF-AT_(c) and/or NFAT-DNA complexin vitro are identified as accessory proteins. Accessory proteins mayalso be identified by crosslinking in vivo with bifunctionalcrosslinking reagents (e.g., dimethylsuberimidate, glutaraldehyde, etc.)and subsequent isolation of crosslinked products that include aNF-AT_(c) polypeptide. For a general discussion of cross-linking, seeKunkel et al. (1981) Mol. Cell. Biochem. 34: 3, which is incorporatedherein by reference. Preferably, the bifunctional crosslinking reagentwill produce crosslinks which may be reversed under specific conditionsafter isolation of the crosslinked complex so as to facilitate isolationof the accessory protein from the NF-AT_(c) polypeptide. Isolation ofcrosslinked complexes that include a NF-AT_(c) polypeptide is preferablyaccomplished by binding an antibody that binds a NF-AT_(c) polypeptidewith an affinity of at least 1×10⁷ M⁻¹ to a population of crosslinkedcomplexes and recovering only those complexes that bind to the antibodywith an affinity of at least 1×10⁷ M⁻¹. Polypeptides that arecrosslinked to a NF-AT_(c) polypeptide are identified as accessoryproteins.

Screening assays can be developed for identifying candidateimmunomodulatory agents as being agents which inhibit binding ofNF-AT_(c) to an accessory protein (e.g. AP-1) under suitable bindingconditions.

Expression of NF-AT_(c) Polypeptides

The nucleic acid sequences of the present invention capable ofultimately expressing the desired NF-AT_(c) polypeptides can be formedfrom a variety of different polynucleotides (genomic or cDNA, RNA,synthetic oligonucleotides, etc.) as well as by a variety of differenttechniques.

As stated previously, the DNA sequences will be expressed in hosts afterthe sequences have been operably linked to (i.e., positioned to ensurethe functioning of) an expression control sequence. These expressionvectors are typically replicable in the host organisms either asepisomes or as an integral part of the host chromosomal DNA. Commonly,expression vectors will contain selection markers, e.g., tetracyclineresistance or hygromycin resistance, to permit detection and/orselection of those cells transformed with the desired DNA sequences(see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

E. coli is one prokaryotic host useful particularly for cloning the DNAsequences of the present invention. Other microbial hosts suitable foruse include bacilli, such as Bacillus subtilis, and otherEnterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(see, Winnacker, “From Genes to Clones,” VCH Publishers, N.Y., N.Y.(1987), which is incorporated herein by reference). Eukaryotic cells areactually preferred, because a number of suitable host cell lines capableof secreting intact human proteins have been developed in the art, andinclude the CHO cell lines, various COS cell lines, HeLa cells, myelomacell lines, Jurkat cells, etc. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter, an enhancer (Queen et al. (1986) Immunol. Rev. 89: 49, whichis incorporated herein by reference), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papillomavirus, and thelike. The vectors containing the DNA segments of interest (e.g.,polypeptides encoding a NF-AT_(c) polypeptide) can be transferred intothe host cell by well-known methods, which vary depending on the type ofcellular host. For example, CaCl transfection is commonly utilized forprokaryotic cells, whereas CaPO₄ treatment or electroporation may beused for other cellular hosts. (See, generally, Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,(1982), which is incorporated herein by reference). Usually, vectors areepisomes and are maintained extrachromosomally.

Expression of recombinant NF-AT_(c) protein in cells, particularly cellsof the lymphopoietic lineage, may be used to identify and isolate genesthat are transcriptionally modulated, either positively or negatively,by the presence of NF-AT_(c) protein. Such genes are typically initiallyidentified as cDNA clones isolated from subtractive cDNA libraries,wherein RNA isolated from cells expressing recombinant NF-AT_(c) and RNAisolated from control cells (i.e., not expressing recombinant NF-AT_(c))are used to generate the subtractive libraries and screening probes. Insuch a manner, NF-AT_(c)-dependent genes may be isolated. NFAT-dependentgenes (or their regulatory sequences operably linked to a reporter gene)may be used as a component of an in vitro transcription assay employinga NF-AT_(c) polypeptide as a necessary component for efficienttranscription; such transcription assays may be used to screen foragents which inhibit NF-AT_(c)-dependent gene transcription and arethereby identified as candidate immunomodulatory agents.

Methods for Forensic Identification

The NF-AT_(c) polynucleotide sequences of the present invention can beused for forensic identification of individual humans, such as foridentification of decedents, determination of paternity, criminalidentification, and the like. For example but not limitation, a DNAsample can be obtained from a person or from a cellular sample (e.g. ,crime scene evidence such as blood, saliva, semen, and the like) andsubjected to RFLP analysis, allele-specific PCR, or PCR cloning andsequencing of the amplification product to determine the structure ofthe NF-AT_(c) gene region. On the basis of the NF-AT_(c) gene structure,the individual from which the sample originated will be identified withrespect to his/her NF-AT_(c) genotype. The NF-AT_(c) genotype may beused alone or in conjuction with other genetic markers to conclusivelyidentify an individual or to rule out the individual as a possibleperpetrator.

In one embodiment, human genomic DNA samples from a population ofindividuals (typically at least 50 persons from various racial origins)are individually aliquoted into reaction vessels (e.g., a well on amicrotitre plate). Each aliquot is digested (incubated) with one or morerestriction enzymes (e.g., EcoRI, HindIII, SmaI, BamHI, SalI, NotI,AccI, ApaI, BglII, XbaI, PstI) under suitable reaction conditions (e.g.,see New England Biolabs 1992 catalog). Corresponding digestion productsfrom each individual are loaded separately on an electrophoretic gel(typically agarose), electrophoresed, blotted to a membrane by Southernblotting, and hybridized with a labeled NF-AT_(c) probe (e.g., afull-length human NF-AT_(c) cDNA sequence of FIG. 1). Restrictionfragments (bands) which are polymorphic among members of the populationare used as a basis to discriminate NF-AT_(c) genotypes and therebyclassify individuals on the basis of their NF-AT_(c) genotype.

Similar categorization of NF-AT_(c) genotypes may be performed bysequencing PCR amplification products from a population of individualsand using sequence polymorphisms to identify alleles (genotypes), andthereby identify or classify individuals.

Yeast Two-Hybrid Screening Assays

Yeast two-hybrid systems may be used to screen a mammalian (typicallyhuman) cDNA expression library, wherein cDNA is fused to a GAL4 DNAbinding domain or activator domain, and a NF-AT_(c) polypeptide sequenceis fused to a GAL4 activator domain or DNA binding domain, respectively.Such a yeast two-hybrid system can screen for cDNAs encoding proteinswhich bind to NF-AT_(c) sequences. For example, a cDNA library can beproduced from mRNA from a human mature T cell line or other suitablecell type. Such a cDNA library cloned in a yeast two-hybrid expressionsystem (Chien et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 9578 orCell 72: 233) can be used to identify cDNAs which encode proteins thatinteract with NF-AT_(c) and thereby produce expression of theGAL4-dependent reporter gene. Polypeptides which interact with NF-AT_(c)can also be identified by immunoprecipitation of NF-AT_(c) with antibodyand identification of co-precipitating species. Further, polypeptidesthat bind NF-AT_(c) can be identified by screening a peptide library(e.g., a bacteriophage peptide display library, a spatially definedVLSIPS peptide array, and the like) with a NF-AT_(c) polypeptide.

Methods for Rational Drug Design

NF-AT_(c) polypeptides, especially those portions which form directcontacts in NF-AT complexes, can be used for rational drug design ofcandidate NFAT-modulating agents (e.g., antineoplastics andimmunomodulators). The substantially purified NF-AT_(c) and theidentification of NF-AT_(c) as a docking partner for AP-1 activities asprovided herein permits production of substantially pure NFATpolypeptide complexes and computational models which can be used forprotein X-ray crystallography or other structure analysis methods, suchas the DOCK program (Kuntz et al. (1982) J. Mol. Biol. 161: 269; Kuntz ID (1992) Science 257: 1078) and variants thereof. Potential therapeuticdrugs may be designed rationally on the basis of structural informationthus provided. In one embodiment, such drugs are designed to preventformation of a NF-AT_(c) polypeptide: AP-1 polypeptide complex. Thus,the present invention may be used to design drugs, including drugs witha capacity to inhibit binding of NF-AT_(c) to form NFAT.

Particularly preferred variants are structural mimetics of a dominantnegative NF-AT_(c) mutants, such as a polypeptide consisting essentiallyof amino acids 1-418 of FIG. 1 and substantially lacking amino acidscarboxy-terminal to residue 418. Such mimetics of dominant-negativemutant polypeptides can have substantial activity as antagonists orpartial agonists of NF-AT activation (and hence T cell activation).

The following examples are offered by way of example and not by way oflimitation. Variations and alternate embodiments will be apparent tothose of skill in the art.

EXPERIMENTAL EXAMPLES

Overview

We have purified two related proteins encoded by separate genes thatrepresent the preexisting or cytosolic components of NF-AT. Expressionof a full length cDNA for one of these proteins, NF-AT_(c), activatesthe IL-2 promoter in non-T lymphocytes, while a dominant negative ofNF-AT_(c) specifically blocks activation of the IL-2 promoter in Tlymphocytes, indicating that NF-AT_(c) is required for IL-2 geneexpression and is responsible for the restricted expression of IL-2.NF-AT_(c) RNA expression is largely restricted to lymphoid tissues andis induced upon cell activation. The second protein, NF-AT_(p), ishighly homologous to NF-AT_(c) over a limited domain, but exhibits widertissue distribution and is highly expressed in tissues characterized byCa++-dependent regulation. Together these proteins are members of a newfamily of DNA binding proteins, which are distantly related to theDorsal/Rel family (Nolan and Baltimore (1992) Current Biology. Ltd. 2:211-220). Agents that increase intracellular Ca++ or that activateprotein kinase C independently produce alterations in the mobility ofNF-AT_(c), indicating that distinct signaling pathways converge onNP-AT_(c) to regulate its function.

Since our previous work indicated that the cytosolic component of NF-ATwas present at relatively low concentrations in human lymphoid celllines (Northrop et al. (1993) J. Biol. Chem. 268: 2917-2923), we choseto purify NF-AT_(c) from bovine thymus. Amino acid sequence, obtainedfrom 6 peptides, was used to isolate two overlapping human cDNA clonesspanning 2742 nucleotides (FIG. 1). The cDNA encodes a protein of 716amino acids with a predicted molecular weight of 77,870. An in-framestop codon upstream from the initiator methionine indicates that theentire NF-AT_(c) protein is encoded by this cDNA. A unique repeatedsequence of 13 residues was also identified. The carboxy-terminal halfof NF-AT_(c) shows limited similarity to the DNA binding anddimerization regions of the Dorsal/Rel family of transcription factors(FIG. 4, for review, Nolan and Baltimore (1992) Current Biology, Ltd. 2:211-220) however, NF-AT_(c) appears to be the most distantly relatedmember of the group. There are a significant number of amino acidchanges resulting in charge reversals between the Rel family members andNF-AT_(c), suggesting that charge might be conserved at these positionsto maintain salt bridges. Six additional peptides obtained from thepurified bovine protein are derived from the bovine homolog ofNF-AT_(p), a cDNA fragment of which was reported by McCaffrey et al.(1993) Science 262: 750-754). Comparison of NF-AT_(c) and NF-AT_(p)reveals that they are products of distinct genes with 73% amino acididentity in the Rel similarity region (FIG. 4), however, there is verylittle similarity outside this region. A murine cDNA for NF-AT_(c) wasisolated and the predicted protein was found to be 87% identical tohuman NF-AT_(c), and distinctly different from murine NF-AT_(p).

Example 1

Determination of the Nucleotide and Amino Acid Sequence of HumanNF-AT_(c) cDNA

This example represents the isolation and purification of this novelhuman NF-AT protein, NF-AT_(c), the determination of the amino acidsequence of its fragments and the isolation and sequencing of the cDNAclone encoding this protein.

The protein was purified from bovine thymus glands obtained from newborncalves. Approximately 20 bovine thymuses were homogenized to make acytosolic extract which was then subjected sequentially to 1) ammoniumsulfate precipitation, 2) sulphopropyl Sepharose chromatography, 3)heparin agarose chromatography, 4) affinity chromatography using amultimerized binding site for NF-AT_(c) with the sequence5′-ACGCCCAAAGAGGAAAATTTGTTTCATACA-3′ (SEQ ID NO:39) coupled to sepharoseCL4B, and 5) HPLC on a reverse phase C4 column. The resulting purifiedprotein was subjected to cleavage with LysC/ArgC and fragments isolatedby HPLC. The sequences of these individual fragments were thendetermined by automated Edman degradation. Sequences obtained included:LRNSDIELRKGETDIGR (SEQ ID NO:35) and LRNADIELR (SEQ ID NO:40).Degenerate oligos corresponding to GETDIG (SEQ ID NO:41) (reverseprimer) and RNADIE (SEQ ID NO:42) (forward primer) were made. Thedegenerate oligo PCR primers had the following sequences:

A forward: (A/C)GIAA(C/T)GCIGA(C/T)AT(A/C/T)GA(A/G) (SEQ ID NO:43) Areverse: ICC(A/G/T)AT(A/G)TCIGT(C/T)TCICC (SEQ ID NO:44)

To isolate the cDNA, oligonucleotide probes were made corresponding tothe determined amino acid sequence and used as PCR primers to isolate a45 base fragment from bovine cDNA prepared from the bovine thymus. Thebovine PCR product comprised the nucleotide sequence CTG CGG AAA whichencodes -L-R-K-. The same 45 bp fragment can be amplified from human andmouse sources.

This bovine PCR product was then used to screen a cDNA library of thehuman Jurkat T cell line. Clones were isolated at frequencies of about 1in 100,000 to 1 in 200,000. A total of five human cDNA clones of variouslengths were isolated. Two overlapping clones, one containing the 5′ endand one containing the 3′ end were ligated together using a unique EcoRIrestriction site present in each clone, to produce a full-length cDNAwhich corresponded in length to the messenger RNA determined by Northernblotting.

The sequence of the NF-AT_(c) cDNA was determined by the Sanger methodand the complete nucleotide and predicted amino acid sequence is shownin FIG. 1. The initiator methionine indicated in FIG. 1 was determinedby fusing this reading frame to a glutathione transferase gene andtransfecting the resultant clone into bacteria. The resultant cloneproduced a fusion protein of the proper molecular weight, indicatingthat the reading frame designated with the initiator methionine isindeed the correct reading frame. The position of the stop codon wasdetermined by a similar procedure. In addition, the stop codoncorresponds to the reading frame for nine of the determined amino acidsequences.

The total NF-AT_(c) protein structure was aligned against individual Relproteins using a MacIntosh shareware program called DOTALIGN utilizingthe alignment parameters of the FASTA programs. Significant homology wasobserved that corresponded to the Rel domains of these proteins.Enhanced amino acid residue alignment was done using ALIGN from the samesuite of programs. Alignment of the Rel similarity regions of NF-AT_(c)and NF-AT_(p) was done by hand with no insertions necessary,, The Miyataalphabet (Miyata et al. (1979) J. Mol. Evol. 12: 214-236) was used todetermine similar residues. FIG. 4 shows results of such sequencealignments.

Example 2

Expression of NF-AT_(c) in T and non-T Cells

The cDNA shown in FIG. 1 was fused to the Hemophilus influenzahemaglutinin (HA) 12 amino acid epitope tag in the determined readingframe and operably linked to the SRα promoter in the vector pBJ5 (Lin etal, 1990, Science 249:677-679). The resultant construct was transientlytransfected by electroporation into Jurkat human T lymphocytes, and intoCos fibroblast cells. Expression of the epitope-tagged NF-AT_(c) proteinwas determined by Western blotting of whole cell extracts prepared fromthe transfected cells, using an antibody (12CA5, Berkeley Antibody Co.,CA) that detects the HA epitope.

FIG. 2 shows that NF-AT_(c) cDNA construct is able to express a proteinof appoximately 120 kDA corresponding precisely in size to that of thepurified protein, in both Jurkat T cells and cos cells (see lanes 3 and6 labeled NF-AT*. Lane 2 shows as control, NF-AT without the epitope tagwhich cannot be detected in the Western blot).

Example 3

Transfection of NF-AT_(c) Activates Transcription in Both Cos and JurkatCells

The NF-AT_(c) cDNA was operably linked to a portion of the SV40 earlygene promoter and the HIV transcription regulatory regions in the pBJvector. This expression vector was co-tranfected into Jurkat and Coscells with either a) three copies of NF-AT binding site linked to anddirecting transcription of luciferase (results shown in FIG. 3A and 3B)the entire IL-2 enhancer/promoter directing transcription of luciferase(results shown in FIG. 3B). Cytosolic extracts were prepared andluciferase assays carried out by standard procedures (de Wet et al,1987, Mol. Cell. Biol. 7:724-837).

The results demonstrate that in both Cos cells and Jurkat cells,overexpression of the NF-AT_(c) protein dramatically enhancesNF-AT-dependent transcription by 50-1000 fold (see FIG. 3A). Inaddition, overexpression of the NF-AT_(c) protein in Cos cells activatesthe IL-2 promoter, which in the absence of NF-AT_(c) cannot otherwise beactivated (see FIG. 3B).

These results indicate that the cDNA clone encodes a functionalNF-AT_(c) protein and that NF-AT_(c) is the protein which restrictsexpression of interleukin-2 to T cells.

Example 4

NF-AT_(c) mRNA and Protein Expression

NF-AT_(c) mRNA is absent in Hela cells (FIG. 5, panel a, lane 7), a cellline incapable of IL-2 or NF-AT-dependent transcription, but isinducible in Jurkat cells (FIG. 5, panel a). This induction is sensitiveto cyclosporin A, (CsA), indicating that NF-AT_(c) may participate in anauto-stimulatory loop as CsA has been shown to block its nuclearassociation (Flanagan et al. (1991) Nature 352: 803-807). Two B celllines, muscle tissue, Hep G2 cells and myeloid leukemia cells do notexpress NF-AT_(c) mRNA (FIG. 5, panel b). These observations areconsistent with the observed T cell-restricted pattern of IL-2transcription and NF-AT activity. Previous studies (Verweij et al.(1990) J. Biol. Chem 265: 15788-15795) revealed NF-AT-dependenttranscription predominantly in spleen, thymus and skin of transgenicmice expressing an NF-AT-dependent reporter gene. Consistent with theseobservations, murine NF-AT_(c) mRNA shows the same pattern of expression(FIG. 5 panel c). Small amounts of NF-AT_(c) expression are seen in lungand heart, however, this may be due to contamination with circulating Tcells. Murine NF-AT_(p) mRNA, also assayed by quantitative ribonucleaseprotection, was found to be expressed at approximately equal levels inbrain, heart, thymus and spleen (FIG. 5, panel c). In contrast toNF-AT_(c), NF-AT_(p) was not inducible by PMA and ionomycin (FIG. 5,panel c).

METHODS. Specific human or mouse NF-AT_(c) or mouse NF-AT_(p) cDNAfragments were used as templates for the synthesis of RNA transcripts.Ribonuclease protection was done according to Melton et al. (1984) Nucl.Acids. Res. 12: 7035-7056) using 10 μg of total RNA. Splenocytes andthymocytes were isolated and treated as described (Verweij et al. (1990)J. Biol. Chem 265: 15788-15795) before isolating RNA, otherwise wholetissue was used.

Example 5

Functional Expression of NF-AT_(c)

NF-AT luciferase and IL-2 luciferase have been described (Northrop etal. (1993) J. Biol. Chem. 268: 2917-2923). β28 luciferase wasconstructed by inserting a trimerized HNF-I recognition site (β28) inplace of the NF-AT recognition sites in NF-AT luciferase. The plasmidpSV2CAT (Gorman et al. (1982) Mol. Cell. Biol. 2: 1044-1050) was used asan internal control for transfection efficiency. Cells were transfectedwith 1.5 ug of luciferase reporter and 3 ug of expression construct asdescribed. After 20 hours of growth, cells were stimulated for 8 hrs.with 20 ng/ml PMA plus or minus 2 uM ionomycin, and harvested forluciferase (de Wet et al. (1987) Mol. Cell. Biol. 7:.725-737) and CATassays (Gorman et al. (1982) Mol. Cell. Biol. 2: 1044-1050).

Cos cells were transfected with epitope tagged NF-AT_(c) as described.Cos cells, Jurkat cells, and murine thymocytes were stimulated for 3 hr.with PMA and ionomycin, Hela cells were stimulated for 3 hr with PMAalone and nuclear extracts prepared as described (Fiefing et al. (1990)Genes & Dev. 4: 1823-1834). Cytosols were prepared from non-stimulatedCos cells. Gel mobility shifts were performed as previously described(Flanagan et al. (1991) Nature 352: 803-807; Northrop et al. (1993.) J.Biol. Chem. 268: 2917-2923). Antisera were raised in mice immunized withbacterially expressed glutathione S-transferase fusion proteins usingthe vector pGEX-3X (Pharmacia) and purified on glutathione agarose.Fusion proteins contained NF-AT_(c) residues 12 to 143 (immune-1) and 12to 699 (immune-2).

NF-AT_(c), expressed in non T cell lines specifically activatedtranscription from the NF-AT site and the IL-2 promoter, (FIG. 6 panel a(left), and FIG. 6 panel b). In transiently transfected Jurkat cells,overexpression of NF-AT_(c) activated an NF-AT-dependent promoter butnot an HNF-1-dependent promoter (FIG. 6 panel a (right)) or anAP-1-dependent promoter. Transfection of the NF-AT_(c) cDNA gives riseto DNA binding activity that is indistinguishable from endogenous NF-AT(FIG. 6 panel c, lanes 1-4). Antibody directed against the RA epitopeencoded by the transfected cDNA induces a supershift of the NF-ATcomplex indicating that NF-AT_(c) participates in this activity. Thenuclear NF-AT activity in transfected Cos cells comigrates with, and hasthe same binding specificity as, the native nuclear complex in T-cells(FIG. 6 panel c, lanes 4-11). Cytosolic extracts from NF-AT_(c),transfected Cos cells can reconstitute NF-AT DNA binding activity whenmixed with Hela nuclear extract (FIG. 6 panel c, lanes 12-16) as docytosolic extracts from T-cells (Flanagan et al. (1991) Nature 352:803-807; Northrop et al. (1993) J. Biol. Chem. 268: 2917-2923). Antiseraraised against bacterially expressed fragments of NF-AT_(c) that have nosimilarity to NF-AT_(p) are able to induce a supershift of theendogenous NF-AT complex, but not the AP-1 complex, from Jurkat cells orthymocytes (immune-1 and immune-2 respectively, FIG. 6 panel d).Immune-2 antisera reduced the DNA-protein complex produced using murinethymic nuclear extracts significantly, consistent with the relativelyequal representation of NF-AT_(c) and NF-AT_(p) peptides in the purifiedprotein from bovine thymus.

Example 6

NF-AT_(c) Dominant Negative Mutant Assayed in Transient TransfectionAssays

A dominant negative NF-AT_(c), prepared after extensive deletionanalysis of the cDNA, indicated that the amino terminal domain wouldblock NF-AT-dependent function without affecting binding. This region ofthe cDNA is not found in NF-AT_(p) and hence can be used to assess thecontribution of NF-AT_(c) to the activation of the IL-2 gene. Thedominant negative NF-AT_(c) used consists of a carboxy terminaltruncation of the epitope tagged NF-AT_(c) expression plasmid (supra)extending to the PvuII site at amino acid 463. Transfection of thisdominant negative resulted in more than 90% inhibition of IL-2 promoterfunction as well as transcription directed by the NF-AT site (FIG. 7).This effect was highly specific since transcription directed by the AP-1site or the RSV promoter and enhancer were relatively unaffected (FIG.7). These results strongly indicate that NF-AT_(c) contributessubstantially to IL-2 gene expression in T cells.

Dominant-negative NF-AT_(c) polypeptides or peptidomimetics thereof canbe used as pharmaceutical antagonists of NF-AT-mediated activation of Tcells. In one variation, such drugs can be used as commercial researchreagents for laboratory testing and analysis of T cell activation andthe like, among many other uses (e.g., immunosuppressant).

Example 7

Post-Translational Modification of NF-AT_(c)

Post-translational modification of NF-AT_(c) was investigated in cellstreated with agents that activate PKC or increase intracellular Ca⁺⁺.Cells were transfected with NF-AT_(c) as described in FIG. 2 andstimulated as shown for 2 hrs plus or minus 100 ng/ml CsA. Whole celllysates were analyzed by western blotting as in FIG. 2. The bulk ofNF-AT_(c) in cells treated with ionomycin migrates faster than that innon-treated cells and this mobility shift is inhibited by CsA (FIG. 8,lanes 1, 3-4). This is consistent with a dephosphorylation event,possibly by direct action of calcineurin (Clipstone and Crabtree (1992)Nature 357: 695-697), however, any of a large number of processes couldproduce the observed mobility changes. There is evidence that NF-AT_(p)is a substrate for calcineurin, however, the mobility shifts produced byphosphatase treatment of NF-AT_(p) or NF-AT_(c) are far greater thanthose observed in FIG. 8. These observations indicate that NF-AT_(c) isnot a direct substrate of calcineurin. PMA treatment produces a slowermigrating NF-AT_(c) (FIG. 8, lane 2); therefore, PKC-activated pathwayslikely contribute to NF-AT activity by modification of NF-AT_(c) inaddition to activation of the nuclear component.

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the claims.

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence which is at least 90% identical to at least 20 consecutiveamino acids of SEQ ID NO: 38, wherein the percent identity is determinedwith the algorithm GAP, BESTFIT, or FASTA in the Wisconsin GeneticsSoftware Package Release 7.0, using default gap weights.
 2. The isolatedpolypeptide of claim 1, comprising an amino acid sequence which is atleast 95% identical to at least 20 consecutive amino acids of SEQ ID NO:38, wherein the percent identity is determined with the algorithm CAP,BESTFIT or FASTA in the Wisconsin Genetics Software Package Release 7.0,using default gap weights.
 3. The isolated polypeptide of claim 2,comprising at least 20 consecutive amino acids of SEQ ID NO:
 38. 4. Theisolated polypeptide of claim 1, wherein the amino acid sequence confersto the polypeptide a biological activity of an NF-AT polypeptide.
 5. Theisolated polypeptide of claim 4, which binds to a nuclear component ofan NF-AT complex.
 6. The isolated polypeptide of claim 4, whichmodulates gene transcription.
 7. The isolated polypeptide of claim 4,which stimulates gene transcription.
 8. The isolated polypeptide ofclaim 4, which decreases gene transcription.
 9. The isolated polypeptideof claim 4, which binds an NF-AT recognition element.
 10. The isolatedpolypeptide of claim 4, which competitively antagonizes NF-AT.
 11. Theisolated polypeptide of claim 10, which is a dominant negative mutant.12. The isolated polypeptide of claim 1, which is a naturally-occurringNF-AT polypeptide.
 13. The isolated polypeptide of claim 12, which is amammalian NF-AT polypeptide.
 14. The isolated polypeptide of claim 13,which is a human NF-AT polypeptide.
 15. The isolated polypeptide ofclaim 1, comprising an amino acid sequence which is at least 90%identical to at least 20 consecutive amino acids of the Rel SimilarityDomain set forth in SEQ ID NO: 51, wherein the percent identity isdetermined with the algorithm GAP, BESTFIT, or FASTA in the WisconsinGenetics Software Package Release 7.0, using default gap weights. 16.The polypeptide of claim 15, comprising an NF-AT amino acid sequence ofat least 20 consecutive amino acids of the Rel Similarity Domain setforth in SEQ ID NO:
 51. 17. The polypeptide of claim 1, which comprisesa Rel Similarity domain having an amino acid sequence which is at leastabout 73% identical to the amino acid sequence set forth in SEQ ID NO:51, wherein the percent identity is determined with the algorithm GAP,BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release7.0, using default gap weights.
 18. The polypeptide of claim 17, whichcomprises a Rel Similarity domain having an amino acid sequence which isat least about 90% identical to the amino acid sequence set forth in SEQID NO: 51, wherein the percent identity is determined with the algorithmGAP, BESTFIT, or FASTA in the Wisconsin Genetics Software PackageRelease 7.0, using default gap weights.
 19. The polypeptide of claim 18,which comprises a Rel Similarity domain set forth in SEQ ID NO:
 51. 20.The isolated polypeptide of claim 2, wherein the amino acid sequenceconfers to the polypeptide a biological activity of an NF-ATpolypeptide.
 21. The isolated polypeptide of claim 20, which binds to anuclear component of an NF-AT complex.
 22. The isolated polypeptide ofclaim 20, which modulates gene transcription.
 23. The isolatedpolypeptide of claim 20, which stimulates gene transcription.
 24. Theisolated polypeptide of claim 20, which decreases gene transcription.25. The isolated polypeptide of claim 20, which binds an NF-ATrecognition element.
 26. The isolated polypeptide of claim 20, whichcompetitively antagonizes NF-AT.
 27. The isolated polypeptide of claim26, which is a dominant negative mutant.
 28. The isolated polypeptide ofclaim 3, wherein the amino acid sequence confers to the polypeptide abiological activity of an NF-AT polypeptide.
 29. The isolatedpolypeptide of claim 28, which binds to a nuclear component of an NF-ATcomplex.
 30. The isolated polypeptide of claim 28, which modulates genetranscription.
 31. The isolated polypeptide of claim 28, whichstimulates gene transcription.
 32. The isolated polypeptide of claim 28,which decreases gene transcription.
 33. The isolated polypeptide ofclaim 28, which binds an NF-AT recognition element.
 34. The isolatedpolypeptide of claim 28, which competitively antagonizes NF-AT.
 35. Theisolated polypeptide of claim 34, which is a dominant negative mutant.36. The polypeptide of claim 1, comprising a polypeptide that isheterologous with respect to the amino acid sequence which is at least90% identical to at least 20 consecutive amino acids of SEQ ID NO: 38.37. The polypeptide of claim 2, comprising a polypeptide that isheterologous with respect to the amino acid sequence which is at least95% identical to at least 20 consecutive amino acids of SEQ ID NO: 38.38. The polypeptide of claim 3, comprising a polypeptide that isheterologous with respect to the 20 consecutive amino acids of SEQ IDNO: 38.